Ultrathin cryo-sectioning,

immuno - gold labeling

A practical introduction

George Posthuma
Viola Oorschot
Janice Griffith
Elly van Donselaar
Despina Xanthakis
Suzanne van Dijk
Corlinda ten Brink

List of abbreviations:
AB:
APD:
BSA:
CV:
EM:
FA:
FCS:
GA:
IgG:
LE:
LN2
MC:
Mwt:
PAG:
PB:
PBS:
PIPES
HEPES
PFA:
PVA:
PVP:
RT:
TA:
UA:

Antibody
Average particle diameter
Bovine serum albumin
Coefficient of variation
Electron microscope
Formaldehyde
Fetal Calf Serum
Glutaraldehyde
Immuno-globulin G
Labeling efficiency
Liquid nitrogen
Methyl cellulose
Molecular weight
Protein A-gold
Phosphate buffer
Phosphate buffered saline
Piperazine-N,N-bis(2-ethanesulfonic acid
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
Paraformaldehyde
Polyvinyl alcohol
Polyvinylpyrrolidon
Room temperature
Tannic acid
Uranyl acetate

Table of Contents
1.
2.
3.
3.1.
3.2.
3.3.
3.4.
3.5.
3.5.1.
3.5.2.
3.5.3.
3.5.4.
3.5.5.
3.6.
4.
4.1.
4.1.1.
4.1.2.
5.
5.1.
5.2.
5.3.
6.
6.1.
6.2.
6.3.
6.3.1.
6.3.2.
6.4.
6.4.1.
6.5.
6.6.
6.6.1.
6.6.2.
7.
7.1.
7.2.
8.
8.1.
8.2.
8.3.
8.4.
8.5.
8.6.
8.7.
8.8.
8.9.
8.10.
8.11.
8.12.

General introduction ......................................................................... 3

The specimen .................................................................................... 5
Chemical fixation .............................................................................. 5
Formaldehyde .................................................................................... 6
Glutaraldehyde................................................................................... 6
Acrolein ............................................................................................ 7
Mixtures of aldehydes ......................................................................... 7
Other conditions affecting fixation quality ............................................... 7
pH / buffers ....................................................................................... 7
Temperature ...................................................................................... 7
Osmolarity......................................................................................... 7
Duration of fixation ............................................................................. 8
Fixation procedures ............................................................................ 8
Storage and shipping of fixed specimens ................................................ 8
Support and cryoprotection .............................................................. 9
Extracellular support ........................................................................... 9
Tissue ............................................................................................... 9
Cells ................................................................................................. 9
Mounting and freezing .................................................................... 11
Specimen carriers ............................................................................. 11
Mounting ......................................................................................... 11
Freezing and storage of frozen specimens ............................................ 11
Sectioning ....................................................................................... 12
The environment .............................................................................. 12
The ultra cryo-microtome .................................................................. 12
The specimen block .......................................................................... 13
Size and shape of a specimen block: Trimming ..................................... 13
The consistency of the block. ............................................................. 14
The knife ......................................................................................... 15
The art of making glass knives. .......................................................... 16
Knife alignment and approaching the block face .................................... 17
Sectioning ....................................................................................... 19
Compression .................................................................................... 20
The operator .................................................................................... 20
Section retrieval (Pick-up) .............................................................. 20
Mounting of LM-sections .................................................................... 22
Storage of thawed cryosections .......................................................... 23
Immuno-labeling. ........................................................................... 23
How many gold particles can be expected? ........................................... 24
Some background information ............................................................ 24
Immuno-labeling strategies ............................................................... 25
Immuno-double labeling .................................................................... 27
The labeling procedure ...................................................................... 27
Background reduction ....................................................................... 28
The immunoreaction ......................................................................... 29
Controls .......................................................................................... 29
Rinsing ........................................................................................... 30
The marker...................................................................................... 30
Stabilization of the immune reaction ................................................... 30
Double labeling procedure ................................................................. 31

8.13.
8.14.
9.
10.
11.
11.1.
11.2.
11.3.
11.4.
11.5.
11.6.
11.7.
11.8.
11.9.
11.10.
11.11.
11.12.
12.
12.1.
12.2.
12.3.
12.4.
12.5.
12.6.
12.7.
12.8.
12.9.
12.10.
12.11.
12.12.
12.13.
12.14.
12.15.
12.16.
12.17.
13.
13.1.
13.2.
13.3.
14.

Section Light Electron Microscopy (SLEM) labeling. ................................ 32

Immuno-fluorescence labeling ............................................................ 32
Contrast and support ...................................................................... 33
Carbon-Formvar coated grids ......................................................... 34
Protocols ........................................................................................ 35
Diamond knife cleaning ..................................................................... 36
Fixation ........................................................................................... 39
Carbon-Formvar coated grids ............................................................. 42
Correlative light electron microscopy (CLEM) ........................................ 45
Gelatin embedding ............................................................................ 47
Immuno-labeling .............................................................................. 50
Ionizer (Static Line II) usage .............................................................. 53
Mounting & Freezing ......................................................................... 54
Loops and hairs preparation ............................................................... 56
Protein A-Gold (Tannic acid procedure) ................................................ 58
Silan coated microscope slides ........................................................... 63
Section Light Electron Microscopy (SLEM) Immuno-labeling ................... 64
Recipes ........................................................................................... 66
BSA 10% (100 ml) ........................................................................... 66
Formaldehyde 16% (500 ml).............................................................. 66
Formvar solution for grids (100 ml) ..................................................... 67
gelatin 2% in 3cm Petri dish (100 ml) .............................................. 68
Gelatin 12% for cell support (100 ml) .................................................. 68
Methyl cellulose 2% (200 ml) ............................................................. 69
Methyl celluloseuranyl acetate pH 4 (10 ml) ...................................... 69
PBS 10X pH 7.3 (2.5 lit.) ................................................................... 70
PBS/glycine 0.02 M ........................................................................... 70
PHEM, 0.2M buffer pH 6.9 (100ml) ...................................................... 71
Phosphate buffer 0.2M pH 7.4 (100 ml) ............................................... 71
Pipes 0.2M buffer pH 7.3 (200ml) ....................................................... 72
PVP - Sucrose pH 10 (20 ml) .............................................................. 72
Sucrose 2.3M (100 ml) ...................................................................... 73
Uranyloxalicacetate PH7 (100 ml) ....................................................... 73
Uranylacetate 4% pH4 (10 ml) .......................................................... 74
Suppliers ......................................................................................... 75
Addendum ...................................................................................... 76
Protein A- G- L affinities .................................................................... 76
Rousselot gelatin .............................................................................. 77
Short incubation schedule. ................................................................. 78
References Tokuyasu technique. .................................................... 79

1.

General introduction

In this course we are primarily concerned with methods for localizing proteins within
cells using high-resolution electron microscopy. This approach is often referred to as
EM immunolocalization, EM immunolabeling, or immuno EM. We are also concerned
about achieving the best possible preservation of cell ultrastructure, and of the
antigens that we will label with antibodies. But before we get into the details of the
course contents, lets take a brief look at how we got to where we are today, and
why we prefer the methods well present in this course.
Most of what we know about the interior ultrastructure of cells comes from
transmission electron microscopy of epoxy resin sections. Unfortunately, these
sections do not work well for EM immunolabeling because the surface of the epoxy
section is generally not permeable enough to gold markers (Stierhof et al., 1989).
Nor is it possible to immunolabel vitreous cryosections because they have to be kept
cold and dry for imaging. While it is possible to do immunolabeling of freeze fracture
replicas (Rash et al., 2004) this method requires specialized equipment that is not
commonly found in most EM labs. Today the two most common methods for EM
immunolabeling are the Tokuyasu technique, and on-section labeling of methacrylate
resin sections. Both methods owe some debt of gratitude to Fernandez-Moran, who
showed many years ago (1952) that processing samples at low temperatures by
cryo-immobilization, cryo-substitution, or progressive lowering of temperature
deydration (PLT technique) improved the preservation of cell ultrastructure.
On-section labeling of methacrylate resin sections evolved mainly in two directions:
the Lowicryls and LR White resins. (Kellenberger et al. 1980; Weibull et al., 1980)
picked up on the PLT idea of Fernandez-Moran and combined that with methacrylate
resin formulations (Lowicryl K4M and HM20) that could be polymerized at low
temperature. ( Newman et al. 1982: Newman and Jasani, 1983)) introduced the LR
White resin and showed that it would accept both
immunoperoxidase/diaminobenzadine (DAB) and colloidal gold markers for
immunolabeling. Initially, the LR White processing did not involve cryoprocessing,
though nowadays it is sometimes used in conjunction with cryo-fixed and cryosubstituted samples, or LR Gold resin is used. While we believe that these on-section
labeling methods, especially with the Lowicryls, are useful in some situations, we
prefer the speed and convenience of the Tokuyasu method, as well as the fact that it
tends to be less damaging to the cells when done properly.
Tokuyasu realized early on
(Tokuyasu, 1976) that
infiltration of fixed samples
with sucrose would prevent
the ice crystal damage that
was such a problem for the
cryofixation methods of that
era. This method also had the
very significant advantage of
not requiring dehydration prior
to sectioning. It is well known,
and has been convincingly
demonstrated (see chapters in
Crang and Komparens, 1988)
that dehydration is one of the
main culprits for producing the
Figure 1.1 :Graphic representation of the standard
fixation
artifacts that have
processing steps in the Tokuyasu technique.
plagued EM studies for years.

Thus, the Tokuyasu technique is mindful of a basic rule of immunolabeling: that the
less you change the sample, the better the chance of recognition by an antibody.
Sucrose does not enter living cells under normal circumstances, but it does not fix
cells either. Thus, the sucrose infiltration step is necessarily preceded by a fixation
step. In the classical Tokuyasu method the sequence of events is as follows: fixation
support sucrose infiltration freezing sectioning retrieval (storage)
immuno-labeling final contrasting and support (Figure 1.1) Here at the Utrecht
University Medical Center we have been using and improving the Tokuyasu technique
for EM immunolabelling since its earliest days. Jan Willem Slot worked with Tokuyasu
in the early seventies in the lab of Jonathan Singer at the University of California,
San Diego, and the first cryo-ultramicrotome was installed here at Utrecht in 1977.
In these early days Slot and his colleagues continued to modify the basic technique,
often in concert with Tokuyasu himself and with Gareth Griffiths who established a
similar facility at the European Molecular Biology Laboratory (EMBL) in Heidelberg,
Germany. Several other technical developments were emerging in parallel that
would have a big impact on improving this technique and making it easier to
perform. Manufacturers were making better cryo-microtomes for one thing, and
there were improvements in diamond knives at about the same time. It is worth
mentioning that the idea of using diamond knives for sectioning was also an idea of
Fernandez-Moran (Fernandez- Moran, 1953).
Another important step forward for EM immunolabeling was the development of
colloidal gold particles. Inspired by the results of Jrgen Roth and colleagues (Roth,
Bendayan et al. 1978) on resin embedded sections, we started to use gold particles
and developed methods to produce uniform gold particles of different sizes (Slot and
Geuze 1985), which is of paramount importance for localization of different antigens
in the same section. The high electron density of the gold markers allowed more
contrast in the sections. With Gareth Griffiths (Griffiths, McDowall et al. 1984)
modification of Tokuyasus adsorption staining method, an elegant staining
procedure was developed that results in a beautiful delineation of membranes and
contrast differences in cytoplasm and nuclei which we still routinely use. In 1995,
Willisa Liou in our lab started to evaluate the damaging effect of the sucrose
solution that had been used routinely for retrieving and thawing of the cryo-section.
The outcome of that study, in which the entire EM group participated, was a
relatively simple modification in the method of cryosection retrieval, but it had a
staggering effect on the preservation of the ultrastructure in the sections (Liou,
Geuze et al. 1996).
These are a few highlights from the many improvements that were introduced during
the last 30 years and that have made the Tokuyasu technique the reliable and
powerful localization tool which it is now (Slot and Geuze 2007). This manual is an
attempt to give an impression of how the technique is routinely performed in our
laboratory. We update it on a regular basis and this would not have been possible
without the valuable help of the people in the lab. The 2011 CMC course manual is
the result of about 210 years of cumulative experience with immuno-electron
microscopy. It is used during practical courses, but it does not pretend to describe
all possible alternatives found in other laboratories, nor do we try to give a complete
overview of the literature. In the first part we describe the method and give some
basic background without being too elaborate. More in depth information can be
found in some excellent books given in the literature section. The second part will
exist of step-by-step protocols with a tips and tricks section.
In the course we also discuss and demonstrate high pressure freezing and freeze
substitution and their possibilities. In 2010 Kent McDonald from the University of
California, Berkeley joined our course as a lecturer and added the part of high

pressure freezing and freeze substitution to this manual and changed the text into
real English for which we are very grateful.
Because many people were expressing interest in the cryo-sectioning and
immunolabeling skills that had been acquired in our group, we started annual
courses in 1999 with the collaboration and support of Leica Microsystems, Vienna.
The course is 10 days long, which is long enough to receive practical, hands-on
training, and we keep the number of students small to ensure that everyone has
adequate opportunity to learn the material. In the past more than 200 people have
completed the course and we hope that they enjoyed it as much as we enjoyed
working with them. We plan to continue this series in the future as a means to
communicate further developments in this field to our colleagues.

Utrecht, 18-6-2012
George Posthuma
Viola Oorschot
Janice Griffith
Elly van Donselaar
Suzanne van Dijk
Despina Xanthakis
Corlinda ten Brink

2.

The specimen

Only the best specimen is good enough for your experiments.In fact the specimen is
the most important item of the entire procedure. When animals are concerned:
gender, stress, drugs, diet, parasites and transgenic changes may affect the quality
of the tissue you are looking at in an unexpected way. In the case of cultured cells
(transient) transfections, the culture conditions, infections (yeast, bacteria,
mycoplasm), drugs, viruses etc. will also affect the quality of what will be seen in the
electron microscope. Do not waste time on suboptimal specimens.

3.

Chemical fixation

The aim of fixation is to maintain the structural integrity of tissues and cells so that
they can be studied in the electron microscope. Immuno-electron microscopy adds
some additional demands:
1. The substances under investigation (antigens) must remain recognizable for
an antibody
2. The antigens should remain at their natural site of occurrence.
3. The antigens should stay (or become) accessible for the immuno-reagents.
Often these demands are in conflict with each other. Preservation of the
(ultra)structure and natural distribution of the antigens need a decent and quick
fixation but that same fixation may mutilate the antigenic site in such a way that the
antibody cannot recognize it. Furthermore it may intensify the matrix compactness
due to cross-linking of cellular components and will hamper penetration of immunoreagents.
Fixation is lethal to cells due to the fact that most proteins stop functioning.
Inevitably the cell becomes leaky for ions and non cross linked (usually low Mwt)
molecules and ions. When cells and intracellular compartments are still surrounded
by a more or less intact membrane some extraction will occur. However when a

frozen section is picked up all the non cross linked molecules are in direct contact
with a buffer solution and will be extracted quickly. In general, antigens attached to
cellular components like membranes or the cytoskeleton will remain in the
cryosections. When working with these naturally bound antigens, mild fixation may
be an advantage since many cytoplasmic components are extracted from the
sections and the accessibility for immuno-reagents will be increased. When
quantitative retention of antigens (especially soluble) is required a strong fixation is
essential.
Some organisms will need special treatment to achieve a good fixation. For instance
yeast has an extremely tough cell wall that needs to be rendered permeable for
fixatives. Other organisms like C. elegans will live happily ever after in fixative and
different measurements like cryo-immobilization and freeze substitution have to be
taken.
The most frequently used fixation agents for immuno-cytochemistry are aldehydes.
These compounds are toxic. They are very reactive and volatile, in particular
acrolein. Therefore it is important to handle them only in a fume-hood with gloves.
A very useful survey the chemistry of fixation can be found in Griffiths book
(Griffiths, Simons et al. 1983)
3.1.
Formaldehyde
Formaldehyde (FA, methanal, O=CH2, Mwt 30) is a gas that easily dissolves in
water. Usually it is sold as a powder which in fact is a polymer (n>8) called
paraformaldehyde. In water it forms also polymers, which can be turned into
monomers with OH- ions and heating to 63C. For Electron microscopy it is of great
importance to work with monomers. Small molecules can easily enter cells and be
there in time to minimize the changes in the dying cell. Formaldehyde has only one
aldehyde group that can react with free amino groups Cross linking occurs in time
due to the formation of methylene bridges. The formation of methylene bridges
between molecules is enhanced by longer fixation times, higher pH (8 - 8.2) and by
using higher FA concentrations (> 5%) (Fox, Johnson et al. 1985). When high
concentrations are used directly on cells or tissue, osmotic events might occur. For
instance the osmolarity of 8% FA in 0.1M phosphate buffer is 1300, which is more
than 4 times the physiological osmolarity. However the effect of osmolarity is limited
because the formaldehyde molecules will react with the cells constituents and hence
contribute less to the osmolarity of the fixation solution. When osmotic events are
observed the initial fixation can be done at low concentrations (2%) of FA, followed
by stepwise increase in concentration. Its low Mwt allows the FA molecule to
penetrate quickly into specimens. It provides a good morphology especially after
prolonged fixation. (8 72 hour). This fixation process is partly reversible and
prolonged storage of tissue in buffers without fixative may result in a loss of content
and structural integrity. In the Tokuyasu technique it used to be difficult to preserve
the ultrastructure in FA-fixed thawed cryosections, but the introduction of new pick
up procedures has improved that substantially. In general FA is a better fixative than
GA for successful immuno-reactions. Extraction of materials and less cross-linking
favour penetration of immuno-reagents and the antigens themselves are less
denaturized.
3.2.
Glutaraldehyde
Glutaraldehyde (GA, pentanedial, O=CH-(CH2)3-CH=O, Mwt 100) has two reactive
aldehyde moieties, which mainly react with lysine residues of proteins and will
crosslink most proteins very efficient, either intra or inter molecular. For electron

microscopy the application of monomeric GA is a prerequisite because long polymers

will not enter cells quickly. Hence always use EM grade GA. The fixation is
irreversible and its penetration is relatively slow. The morphology is very well
preserved. GA is a strong fixative in immuno-cytochemistry. The ultrastructure is
stably preserved, but antigen denaturation often occurs. During fixation a good
buffering system should be used, otherwise the pH might be lowered 2-3 pH units.
3.3.
Acrolein
Acrolein (propenal, O=CH-CH=CH2, Mwt 56) has one reactive aldehyde group that
mainly reacts with lysine. Furthermore the double bond between the carbon atoms
may play a role in cross linking. Its fixation is irreversible and its penetration into
tissues and cells is relatively fast. Acrolein is mostly used in combination with GA or
FA.
3.4.

Mixtures of aldehydes

A mixture of GA and FA and or acrolein can be used in various combinations. The

effectiveness of such mixtures is not an addition of reactivity of both components.
Both aldehydes will compete for amino groups. Therefore the mixture may result in
stronger fixation than from 2% FA, but cross-linking may be less than when only
0.2% GA is used
3.5.

Other conditions affecting fixation quality

3.5.1.
pH / buffers
The pH should be maintained within a physiological range during the fixation
process. It is known that the aldehyde fixatives lower the pH when they react.
Therefore the buffering capacity of the fixative solution should be strong. Omit PBS
and use a stronger buffer system like 0.1M phosphate buffer. Some commonly used
buffers like TRIS may be less suitable during fixation. Our concern is that the amino
groups will quench the aldehyde action. Furthermore substances can be added to the
buffers that favor the preservation of specific cellular components like cytoskeleton
elements (PHEM buffer). Extra precautions should be taken when the specimen has
been fixed with uranium salts like uranyl acetate, which is often used in freeze
substitution. These salts react with constituents of phosphate buffer and give rise to
unwanted precipitations. A HEPES or Pipes buffer should be used in these cases..
Suitable buffers are phosphate buffer (0.1M, pH 7.4), HEPES (0.1M, pH 7.4) or PIPES
(0.1M, pH 7.4).
3.5.2.
Temperature
The initial fixation should be performed at room temperature or even at 37C. At
those temperatures the structural integrity of the cytoskeleton is better preserved.
Furthermore the diffusion of the fixative into the tissue or cells is faster and the
actual cross-linking will be more effective. After the initial fixation the specimens can
be stored for longer periods of time at 4 C.
3.5.3.
Osmolarity
A physiological osmolarity for a fixative would be 360 mOsm. Some fixatives like FA
can increase the osmolarity to a great extent. For example 8% FA in a 0,1M
phosphate buffer has an osmolarity of 1300 mOsm. However the effect on cells is
7

not as profound as these figures may suggest. First, the aldehydes are presumably
uncharged when added to the cells and therefore they can pass freely through
biological membranes. Next they start to react rather quick with amino moieties and
therefore do not contribute as much as anticipated based on the molarities to the
final osmolarity. However when rather slow reacting substances are used like FA,
osmolarity might be taken into account (see also 3.1).
3.5.4.
Duration of fixation
The time during which specimens should be fixed depends on the size of the
specimen, the nature of the antigen and the used fixative, its concentration and the
temperature. Usually, 2 hr at room temperature is sufficient for fixation with GA
containing fixatives. FA (without GA or acrolein) fixation is usually performed
overnight. Prolonged fixation does not necessarily improve the ultrastructural
preservation, but may affect the antigenicity of antigens under investigation.
Furthermore prolonged fixation may harden the specimen and will affect the
sectioning properties.
3.5.5.
Fixation procedures
It is important to disturb the material to be fixed as little as possible prior to fixation.
For instance do not wash or trypsinize cells when it is not necessary. In particularly
ice cold media or even PBS will influence the cells morphology. It is usually better to
leave them under growing conditions and add fixative. Also the fixative should be at
temperatures of 20 -37 C, at least at the beginning.
Tissues from animals should be fixed by whole body perfusion when possible. After
an initial flush with PBS (RT) to remove the blood, the fixative (RT) is introduced into
the (anesthetized of) animal via an artery or the left ventricle of the heart. Biopsies
should be immersion fixed as soon as they are excised from the body and cut into
thin (<1mm thick) slices in fixative.
3.6.
Storage and shipping of fixed specimens
GA fixed specimens can be stored in buffer for months at 4C. FA fixed specimens
are stored in 1% FA (in buffer) at 4C because of the reversibility of the reaction.
When a combination of GA and FA is used we store the specimens in 1% FA at 4C.
We have been using specimens for more than 5 year without noticeable changes in
ultra-structure and immune reactivity.
Airway regulations consider <8% FA harmless and you are allowed send material in
2% FA wit out special permits. Leakage has to be prevented at all time to prevent
drying of the specimens. Preferentially use culture flaks which are entirely filled with
2% FA in buffer and well-sealed. Petridishes can be sealed with Parafilm, but this is
less safe. During shipment by air, the fixed sample may freeze and ice crystals can
cause substantial damage to the ultrastructure, especially when just formaldehyde
has been used for fixation. To reduce this risk, it is a possibility to ship the
specimens in >1.8M sucrose, to which 1% FA is added. Sometimes the tissues are
sent together with immuno-reagents. Make sure that they are well separated from
each other in different Falcon tubes.

4.

Support and cryoprotection

4.1.
Extracellular support
Embedding of specimens in a matrix similar to the specimen serves several
purposes:
1) Cell suspensions can be concentrated at will and the blocks are easy to
prepare and handle.
2) It allows orientation of specimens (correlative LM-EM)
3) It improves the conditions for sectioning substantially: it fills extracellular
spaces thereby equalizing the consistency of cells and extra cellular spaces.
4) In sections of non embedded cell suspensions and to lesser extent in loose
tissues without embedding cell profiles float around individually when thawed
on the pick up solution. When embedded in a meshwork they behave more
coherently, which is particularly important for serial sectioning or correlative
light and immuno-electron microscopy.
In general we use gelatin as supporting matrix. Gelatin comes in many flavours.
The source (pig-, cow-, fish- etc. bones or skin) and the way it was treated and
purified determine the stiffness of the final gel. Usually this is expressed as Bloom
figure: the higher the Bloom figure the stiffer the gel will be (when the same
percentage is used). The relatively high mol weight of the denaturised collagen
prevents it from entering fixed cells. We use food quality gelatin, which is compatible
with all the buffer systems we have tested till now. A good alternative for gelatin is
low melting point agarose, especially when part of the specimens has to be osmium
fixed and embedded in a resin. After sectioning gelatin can be removed at 37 from
the section while agarose or fixed albumin cannot.
4.1.1.
Tissue
In figure 4.1 a schematic
drawing is made of a simple
way to prepare appropriate
blocks of gelatin supported
tissue. For tissue a microscope
slide is completely covered
with a layer of Parafilm,
whereas on either end an
additional 6 layers of Parafilm
are placed. The specimen (in
37 gelatin) is placed on the
Parafilm and another with
Parafilm covered slide is
placed on top (Figure 4.1-1).
Next this is placed on ice and
the gelatin is allowed to
solidify (Figure 4.1-2). The top
Figure 4.1: embedding of tissue in gelatin
slide with Parafilm is removed
and the gelatin slab with tissue
can be cut in appropriate blocks (Figure 4.1-3,4). Next the block are transferred to
2.3 m sucrose, mounted on a specimen holder (Figure 4.2-5, 6,7) and frozen.
4.1.2.
Cells
Cells are rinsed twice with 37 gelatin and spun down in an Eppendorf tube (Figure
4.2). The tube is placed in ice and the gelatin is allowed to solidify. The bottom of

the tube containing a high concentration of cells in gelatin is cut off, removed from
the bottom of the Eppendorf
tube and cut into 0.5x0.5x 0.5
mm blocks with a razor blade
(Figure 4.2-1,2,3,4). Next the
blocks are infused with sucrose
and mounted on a specimen
holder (Figure 4.1-5,6,7) and
frozen. Cryoprotection
It is important to prevent ice
crystal formation in the
specimens during freezing.
First of all ice crystals would
damage the ultrastructure, but
besides that, good cryosections
can only be cut from vitreous
frozen blocks. This makes
freezing of fresh biological
material a real problem. Ice
crystal formation can only be
prevented when freezing
occurs within milliseconds.
Figure 4.2: Embedding of cells in gelatin, sucrose
Heat conductance becomes the
infiltration and mounting.
limiting factor with the result
that in practice vitreous freezing is restricted to the very surface layer of, at best,
~10 m. This can be extended to a few hundred microns under high pressure
conditions.
Working with chemically fixed tissue, as we do in the Tokuyasu technique, ice crystal
formation is simply circumvented by infusing sucrose into the tissue. Sucrose is a
rather inert, hydrophilic compound that easily diffuses through the cellular
membranes after fixation. It does not seem to affect the fixed tissue, even not at the
highest possible concentration of 2.6M. Tokuyasu introduced the sucrose infusion
and later it was shown by Griffiths et al. (Griffiths, McDowall et al. 1984); and
McDowall et al.(McDowall, Chang et al. 1983) that sucrose solutions of 1.8M or
higher vitrify when they freeze, no matter how slow the freezing takes place. The
entire specimen needs to be infiltrated with 2.3 M sucrose otherwise sectioning
becomes very difficult and ice crystal damage may occur. We use an overnight
infiltration in a rotating carousel at 4 C. In sucrose infused tissue, the cellular
constituents and the extra cellular gelatin probably also contribute to the vitrification
hence lower sucrose concentrations may be sufficient for proper cryoprotection. This
can explain why Tokuyasu had good results with regard to cryoprotection when he
used sucrose concentrations below 1.8M. He recommended initially 0.6 - 1.5M for
different tissues, depending upon their protein density.
Sucrose infusion serves other purposes as well. Around 1980 we found that infusion
of the tissue blocks with 2.3M sucrose improved the sectioning characteristics
substantially (Geuze and Slot 1980) for any tissue and cell preparation that we
worked with. This high concentration of sucrose renders a plasticity to the frozen
blocks that, allows cutting of very thin sections that are still flat and glossy at -120.
Another possibility to improve sectioning is the usage of a mixture of
polyvinylpyrrolidone (PVP) and sucrose especially for loose tissues , cells with large
voids (Tokuyasu 1989) and cells grown on filters.

10

5.

Mounting and freezing

5.1.
Specimen carriers
The aluminum specimen carriers (pins) should fit perfectly in the microtome
otherwise the sectioning will be very irregular. Usually they are provided with the
microtome by the manufacturer; however ordinary rivets seem to perform just as
well and are much cheaper. The sucrose infused block should be glued with sucrose
to the pin. The top of the pin is roughened, with a sharp metal point. The same can
be achieved with sandpaper, but the grains will damage the diamond knife when
they are not removed properly. The small groves improve the attachment of the
specimen to the surface. The surface needs to be clean, hence we sonicate them in
acetone to remove any greasy material and metal remnants (do not touch the
surface of the pins with your fingers!!) Check with binoculars whether the pins are
perfectly clean and free of metal remnants. Again such remnants may ruin a precious
diamond knife. The pins can be reused after removal of the specimens and sucrose
by sonicating them in distilled water and acetone respectively.
5.2.
Mounting
The specimen blocks are removed one by one from the 2.3M sucrose by means of
forceps or a tiny wire loop and placed immediately on the table of a clean pin. Be
careful not to touch the table surface with anything except the specimen, in
particular not with your fingers. Remove most of the excess sucrose with a piece of
filter paper, but leave enough to glue the block at the basal side to the specimen
holder (Figure 4.2-7) Mount the specimen in a good position for sectioning.
Sometimes the specimen has to be sectioned in a specific orientation, for instance
cross sections of muscle fibers or kidney medullar tubules. It may be advisable in
that case to shape the blocks in such a way that orientation still can be recognized,
for instance a long edge parallel to the muscle fibers. The final trimming of the
blocks can then be done right before the mounting, so that you still remember which
side of the block has to be the cutting face. The mounting procedure should be done
as quickly as possible. Water evaporates from the specimen during air exposure, and
then sucrose concentration becomes so high that sucrose crystals will be formed. For
that reason we perform these steps under a binocular in the cold room. If there is no
cold room available, keep the blocks cool and put the pins in a plastic block (not
metal, because you will get water from the air on your pin) in ice and and freeze
your blocks one by one.
5.3.
Freezing and storage of frozen specimens
The mounted blocks, glued by the remaining sucrose to the specimen holder, are
then put in LN2 or in the cryochamber of the ultra cryo-microtome. The sucrose
needs to remain clear after freezing. If it turns milky the sucrose concentration is too
low and water crystals have formed. This will affect the sectioning properties of your
specimen If the crystals have not damaged your specimen, renewed infiltration with
real 2.3 M for 4 hr on a rotator will solve this problem.
When the specimens are relatively big, copper or, to a lesser extent, aluminum
specimen holders cool down much quicker than the specimens and crevasses may
emerge between the holder and the specimen. This will not occur when the specimen
and pin are frozen slowly by freezing them in the (at least 80 C) cryo-chamber of
the microtome. Crevasses will also emerge when the table of the pin has not been
11

cleaned properly (grease). As a result during sectioning chattering, irregular

sectioning will occur, in the worst case the specimen may break away from the pin.
When this happens it is possible to remount the specimen, since freezing is no longer
a crucial step. Pick up the specimen and put it back in 2.3 M sucrose (4 C), leave it
there for 30-60 min and remount.
We use Sanbio cryotubes for storage, its lid fitted with two holes. The holes in the lid
prevent internal pressure build up and form also a grip for firm tweezers when
opening or closing the cryotubes. Each cryotubes contains an aluminium block,
which prevents it from floating. Seven 8 mm holes, drilled in the metal block form
seven numbered compartments, each of which can contain several pins with
specimen blocks, we often use colour coding (using a standard felt pen) for the pins
as well. In this way many specimen blocks can be stored in one LN2 container.
Frozen specimens (on specimen holders) can be stored in LN2 for months or
even years. Prolonged storage in LN2 may freeze-dry the specimen surface to a
certain extent. If this renders sectioning more difficult, renewed sucrose infiltration
and remounting the specimen solves this potential problem.

6.

Sectioning

The quality of ultrathin (cryo) sections is depending many factors:

1. The environment
2. The ultra cryo-microtome
3. The specimen bloc
4. The knife.
5. The Tools
6. The operator
6.1.
The environment
The environment in which the microtome is placed is of great importance to the way
it will function. It should be in a vibration free room without large air flows with a
controlled humidity. Most of the vibration problems are solved when the microtome
is placed on a standard table that comes with the microtome. If this is not sufficient
the microtome can be placed on an active anti vibration device. Air that flows around
the cryochamber will disturb the stability of the atmosphere inside the chamber and
this will result in temperature changes and excessive ice formation in and around the
cryobox. Humidity in the room is a factor that affects the buildup of ice and the static
conditions in the cryochamber. For the Tokuyasu technique a humidity of
approximately 50% is good. When frozen sections from vitrified specimens (without
2.3 M sucrose) are cut for cryo-electron microscopy the humidity needs to be as low
as possible and a special box around the microtome is required.

6.2.
The ultra cryo-microtome
The current ultra-microtomes have reached a technical degree of perfection, that
allow regular feed with an accuracy of 1 nm, which means that 15-200 nm sections
have become reality. Furthermore the sectioning speed can be controlled very
precise from 0.1 mm/sec to 100 mm/sec. The combination of this extremely
accurate and precise movement with precise temperature regulation in the
cryochamber of specimen, knife and cooling gas makes cryo-sectioning a much
12

easier job as compared to 10 years ago. Despite this degree of perfection

maintenance is very important. Spindles and motors ware off and greases do age.
We have our microtomes serviced once a year thoroughly and give them a quick
checkup 6 months later. Special attention should be given to the LN2 pump. When
the pumping is not regular, e.g. the pump is always pumping with the same speed
during operation, the sectioning will be irregular. Before you start sectioning always
check whether the flow and pressure are normal. We judge that by turning the pump
on without the tube toe microtome attached to the pump. If this is OK, but still the
amount of LN2 flowing into the chamber is not sufficient, the tubing is probably
clogged with ice and needs to be cleaned. We keep the pump during the week in the
LN2 and dry it during the weekend. When drying is not sufficient the pumping
mechanism requires cleaning. Last but not least the dewar needs attention: in 3-6
months there will be a lot of ice crystals floating in the LN2, these will eventually
stop the pump from functioning properly.
6.3.
The specimen block
The way a knife interacts with a specimen depends on the size, shape and
consistency of a block.
6.3.1.

Size and shape of a specimen block: Trimming

When you start to cut ultrathin
sections the block needs to be
flat on the front side, small
enough and have a rectangular
(square) form with straight
edges.

Figure 6.1: trimming of a specimen block. A: with a

glass knife, B: with a diamond trimming tool.

13

First the front is flattened using

a good glass knife or a
diamond trimming tool. At high
temp (-100C) with a high
speed (100 mm/sec) relatively
thick sections of 200 300 nm
can be cut. The largest
protrusions should be on the
lower side of the block,
otherwise during the sectioning
a large piece of your block may
chip away (Figure 6.1-A, D).
The total surface should be like
a tiny mirror. White dots in the
section indicate the presence
of plain sucrose and will affect
the sectioning. During the
trimming put the ionizer close
to your knife edge at 9, it will
blow away most of the dust
you generate. At this point a
semi-thin section should be

picked up, stained and evaluated in the light microscope (correct region in the tissue,
enough cells ? etc).
After the area of interest has been selected, the size of the block is determined. It is
possible to cut ultrathin sections of large blocks (2x2 mm) but keep in mind that with
an increasing size of the specimen block the forces necessary to actually make the
section become larger and the sectioning becomes more troublesome. As a general
rule: the smaller the block the easier it is to section. When sections become thinner
the necessity for smaller dimensions becomes more evident. (Al-Amoudi, Dubochet
et al. 2003; Matzelle, Gnaegi
et al. 2003). With the latest
microtome models the block
size can be measured. Usually
sizes of 200-500 m work quit
well.
The shape of a block is also
important for the final result.
For resin sections usually a
trapezoid form is used. This
Figure 6.2: different specimen block forms. A: common
for resin sectioning, B and C: suitable for cryosectioning,
allows an obvious distinction
D: less suitable for cryosectioning.
between subsequent sections
and makes them easy to
separate. Frozen sections are cut dry and have more interactions with the knife
surface. For cryosectioning a square (Figure 6.2-B) or a rectangular (Figure 6.2-C)
block is much more practical. In block no C the long side is 1.5 times longer than
the short side which makes it easy to judge the degree of compression of the
section. With 30% compression the final result is a square section. Avoid an
orientation as shown in Figure 6.2-D.
The sides need to be straight and the resulting block needs to be rectangular. This
can be realized by using a diamond trimming tool, the corner of a glass knife or a
razor blade (and a steady hand) . Trimming with a diamond trimming tool or a glass
knife is quite similar.
A diamond trimming tool (we prefer a 45 cutting angle with 20 sides) cuts both at
the front and at the sides, so it will give straight edges. With a trimming tool one
side is trimmed 50-100 m deep, the knife is retracted, the tool moved to the other
side of the block and again 50-100 m deep is trimmed away. Be aware of the angle
of a trimming tool: when 50 m deep has been removed with a tool with 20 angle
on the sides it also cuts away 18 m of the front of the block. Next the block is
turned 90 and the same routine is performed. Turn the specimen back to its original
position and remove any debris from the surface by cutting one or two 50 nm
sections. With a glass knife only one side can be used and it is necessary to turn the
specimen 4 times 90.

6.3.2.
The consistency of the block.
Extremely soft blocks are very difficult to cut. During the sectioning process these
will have extreme compression which results in a much thicker section than the feed
14

indicates (see Figure 6.3 ).

Hard (brittle) materials are
often also difficult to cut
because chipping will easily
occur as indicated in the lower
panel of Figure 6.3. In
cryosectioning the hardness of
the block can be controlled
with the temperature: the
lower the temperature the
harder the block. Last but not
least the uniformity of the
consistency is also affecting
the sectioning characteristics
of a block. The more
differences in consistency the
Figure 6.3:Sectioning properties of soft (upper panel)
more difficult it is to section,
and brittle (lower panel) specimens. Soft specimens will
especially when soft and hard
yield more compression, while brittle specimens will
portions are parallel to the
have many crevasses or even chip away during
knife edge. Hence embedding
sectioning.
in gelatin for cryosectioning will
improve the sectioning. In contrast to resins, gelatin will not enter the interior of a
cell, so when there are large voids in the specimen, that can not be reached by
gelatin, it might be advantageous to add in addition to the sucrose a high molecular
weight substance like polyvinylpyrrolidon to improve the consistency after freezing.

6.4.
The knife
A knife has a number of given characteristics. First of all there is the material the
knife is made of. For electron microscopy only two materials have been used
routinely to produce ultrathin sections: glass
and diamond. A glass knife is made out of a
special kind of glass with a knifemaker,
usually with an angle of approximately 45
Diamond knives are prepared from natural
gem quality (or even better) diamonds and
come in many sizes and shapes. The top
angle can be as sharp as 15, however those
knives are very vulnerable. The sharper the
knife angle the less compression a knife will
give (Matzelle, Gnaegi et al. 2003; AlAmoudi, Studer et al. 2005) Further more the
radius (Figure 6.4 R)of the very edge of the
Figure 6.4: Knife edge properties: The facet
of a knife is grinded with a certain angle in this
case 35and has a top radius (R) . Both
determine the sharpness of a knife. The way
the surface is treated after grinding
determines the he properties of its surface.

15

knife will also play an important role during sectioning. A blunt knife (large radius)
will never cut. Moreover the surface of the knife that is in contact with the section is
extremely important. The surface of a glass knife is obviously glass, whereas the
surface of a diamond knife is one of the best kept secrets of the diamond knife
manufacturers. After grinding a diamond to the desired dimensions, the polishing
material is removed and the surface is treated in such a way that it gets its final
properties. The message is that for each specific application there is an appropriate
surface available. Especially when the sectioning is performed dry, the surface of the
diamond determines whether sectioning will be a success or not. For cryosectioning
of 2.3 M sucrose infused specimens an almost ideal surface has been created by
Diatome . To use the maximum benefits of such a surface good cleaning is
imperative
6.4.1.
The art of making
glass knives.
The preparation of a glass
knife starts by selecting good
glass strips from which they
are made. The glass should
be tough and preferably
without consistency
differences in the glass
(Pittsburgh glass). When a
new batch of glass strips
arrives, the dimensions and
quality should be carefully
checked. The width of the
strips must be constant
without distortions on the
lateral sides of the strips. If
the strips do not meet your
requirements send them back
to the manufacturer.
The perfect glass knife has a
straight or slightly concave
cutting edge. Its top angle is
approximately 45 and does
not show any serrations in a
binocular. In the 45 plane
the breaking lines are barely
visible.
Figure 6.5-A: Braking of glass knives according to the
balanced brake method). B: The final scratch is
positioned as close as possible to the diagonal of the
square and is broken, this will yield an approximately
45 knife. C: When the scratch is not on the diagonal
the top angle of the knife will be close to 80

16

The best way to make knives

is according to Tokuyasus
balanced break method
(Tokuyasu and Okamura
1959) using a Leica knife
maker (KMR3). In theory a

perfect knife is made by breaking a glass rod in two equal parts, next each part is
broken in two equal parts etc., till squares of 2.5 x 2.5 cm are produced (Figure 6.5A). Because equal forces have been applied to the glass this balanced breaking
method yields squares, with freshly straight edged surfaces. The sharpest knives will
be obtained when the breaking occurs almost exactly on the diagonal of the square
(Figure 6.5-B). Glass will break towards the nearest surface an when the scratch is
away from the diagonal (Figure 6.5-C) the top angle will be close to 80 (!), which
will make sectioning a kind of scraping. Furthermore the crack should develop slowly
using moderate forces and should be perpendicular to the upper surface of the
square. Fast breaking will result in an elevated stress line that will affect the knife
edge in a negative way (Figure
6.5-A). To meet all these
requirements the midpoints of
the upper pins of the knife
maker must necessarily be
positioned at equal distances
from the scoring line. The
support pins at the lower
surface of the glass should be
positioned exactly beneath the
scoring line (Figure 6.6-A). This
can be checked by breaking
several twin blocks. If the
breaking plane shows the
same aberration repeatedly,
the position of the scratch
should be adjusted since the
Figure 6.6: Knife dimensions after braking. A: the stress
position of the support pins is
line (arrow) will be very prominent after fast braking as
set by the manufacturer. To be
shown in the drawings of the upper part of the knife.
able to fracture the squares
B1:Knife with a straight edge, B2: knife with a concave
along the diagonal the position
edge, C3:knife with a convex edge. The latter is not
suitable for sectioning.
of the square is carefully
examined after each fracture and adjusted when necessary. If the counter piece is
hardly visible and the sharp edge is made out of the freshly broken plane, the knife
is probably quite good (Figure 6.6-B, always check with a binocular). With older
knifemakers positioning of the square needs some extra attention and adjustments.
Knives with a convex edge (Figure 6.6-B3) or with elevated stress lines (Figure 6.6-A
fast) should not be used.
Preparing knives as described above may seem laborious, but the invested time is
not idly spent, since the ideal knife will save much time and annoyance during
sectioning. In our lab we store the knives in a plastic box for several months.
6.5.
Knife alignment and approaching the block face
Once the block has been trimmed, the trimming knife is replaced by a sectioning
knife. This should be at sectioning temperature, absolutely clean and without any
frost. If the block is still in the microtome upper and lower part of the block will have
equal distances to the knife edge (alignment north-south). When a previously

17

trimmed or sectioned block is used this may not be the case as shown in figure 6-7 A
and B were the backlight illumination is used to visualize the gap between knife and
specimen. When the reflection on the specimens surface is identical on lower and
upper side of the specimen, there is no need for correction otherwise the front

Figure 6.7: Knife alignment NorthSouth. When the specimen surface is

not equidistant to the knife edge,
while moving up and down, the
reflection on the specimen surface
changes from wide to small. In a
cryo-microtome this cannot be
corrected, so a new blockface needs
to be prepared

Figure 6.8 : Knife alignment

East-West. When the blockface
is not parallel to the knife edge
in east west direction, the knife
must be (very carefully) turned

Figure 6.9 Aproach: When the

backlight is turned on and the knife is
in the vicinity of the trimmed face a
bright white area can be observed on
the block face. This is the reflected
light from the back of the knife and
the specimen surfce(A). When the
knife approaches this reflection
becomes smaller (B) and finally it
changes to a color (C)

18

needs to be equalized by cutting a few sections on a trimming knife since a cryoultra microtome does not have a segment arch. Using previously trimmed block the
lower edge of the block needs to be aligned with the knife edge. After trimming the
edge of the sectioning knife usually is not parallel to the specimen. When the
reflection of the backlight on the block is not perfect rectangular as in Figure 6.8A&C, the knife should be rotated in such a way that the reflection is a perfect
rectangle (Figure 6.8-B).
When block face and knife edge are perfectly aligned the knife needs to be
positioned so close to the block that the next stroke of the microtome will result in a
section. Again the back light is very useful. While moving closer and closer to the
block the reflection becomes smaller and smaller (Figure 6.9 A >B) till it is no longer
white and becomes colored (Figure 6.9-C). Once blue only one or two 50 nm steps
forward are necessary to cut the first section.
6.6.
Sectioning
The ideal section has a perfectly flat surface, has a uniform thickness, no knife
marks, is not folded and has no compression. As with many things such perfection
only exists in fairy tales. When the knife encounters the specimen no one knows
exactly what happens. However the knife has a finite sharpness which means that it
starts to push into the specimen thus cutting a section. Depending on the hardness
of the specimen (Figure 6.3 and Figure 6.11) some block deformation will occur. The
section will be pushed onto the surface of the knife and will be compressed due to
the friction and in a dry environment charge will build up. In reality the initial section
thickness as defined by the feed (T1 in Figure 6.11) will be up to 3 times more due
to these processes (T2 in Figure 6.11). In resin sectioning this also occurs, however
the friction and static problems can be minimized by using a trough with water that
acts as a lubricant and also prevents static charges to build up. In cryo-sectioning

Figure 6.10: Charges on a knife surface

can be regulated with an ionizer.

Figure 6.11 Interactions between knife and specimen.

During sectioning the knife cleaves the specimen. The
lower surface of the section will encounter friction on
the knife surface and compression will occur further
more static charges (--) will be generated

19

many attempt have been made in the past to find something equal to water. Thus
far without success. A fluid has not been found yet, that remains liquid at low
temperature and has the same surface tension as water, a prerequisite for obtaining
a floating section. In cryosectioning a different lubricant is used( Figure 6.10 : --on knife and section). During sectioning static charges build up on the knifes
surface. The amount of charge depends on the weather (humidity) the person
(clothing) the kind of specimen and most important the knifes surface. Till the
introduction of the controllable ionizer this static charges could not be regulated and
you were lucky if everything worked. With an ionizer the amount of charge can be
carefully controlled (in most cases) and the charge on the knife and section make
the section hoover above the knife surface thus reducing compression to a
reasonable level (30%) and allowing the section to be manipulated using an eye
lash. With the correct settings and a bit of luck, it is possible to make long ribbons till
even 2 cm long!
The sectioning speed is also an important factor. In the past the text on the box of a
diamond knife stated: tested at 1mm/sec and that speed was what most people
used. The combination of the blocks consistency, the knife, the charges and the
speed will ultimately determine whether the sectioning will be satisfactory or not. It
is worthwhile to vary the speed when the sectioning does not work. We have varied
in the past between 0.2 mm/s till 80 mm/s! It will not damage your knife and might
be the solution to your sectioning problem.

6.6.1.
Compression
Compression is expressed as: 100- (section length/block surface length)*100 . (i.e.
a percentage). During the sectioning the section pressed onto the knife surface, thus
compressing the section. When the angle between knife surface and block face is
larger, this effect will increase. The second factor is Figure 6.11). The section sticks
to the knifes surface, whereas at the same time it is pushed away from the knife
edge along the knife surface by the rest of the section. The first problem can be
minimized by using knives with a small top angle (Al Ahmoudi et al , 2003), the
second by toning the knife surface with an ionizer and varying cutting speeds.
Unfortunately in cryosectioning compression cannot be avoided.
6.6.2.
The operator
The operator is a very important factor: you need to get acquainted with your
machine in combination with your specimens, knives, environmental circumstances
etc. Besides skills, the person hem- (or her-) self influences the sectioning because
the operator acts as a source or drain of static electricity. Well insulating shoes in
combination with synthetic fabrics will render sectioning virtually impossible. We
advice cotton fabrics and sometimes a grounding bracelet (like people that handle
electronic components).

7.

Section retrieval (Pick-up)

Once the cryo ultra-microtome produces relatively flat, minimally compressed

sections, they have to be retrieved from the knife. Under the right conditions modern
20

cryo ultra-microtomes, like the Leica Ultracut S or T/FCS and more recently the
UC7/FC7, regularly produce ribbons of sections. Usually the sections are guided
with an eyelash on top of a wooden stick away from the knife edge. The ribbons are
detached from the knife edge and moved aside somewhere down on the diamond
knife or on the epoxy between knife holder and diamond knife (NB: do not transfer
to the metal part of the knife, the sections cannot be retrieved properly) The size of
the ribbons should not exceed half the diameter of the wire loop used to retrieve the
sections. Sections have a tendency to jump towards the pickup solution when the
solution temperature is not correct. During their journey to the pickup solution
sections wrinkle. Therefore it is advisable to aim for the center part of the drop, to
prevent this as much as possible. When 2.3 M sucrose is used sections need some
space to spread out over the pick-up drop during thawing. Usually not more than 4
or 5 sections should be included.
When enough sections are collected, stop the sectioning and move them away from
the knife edge and pick them up. The ionizer should be turned off during section
retrieval. A wooden stick with a stainless steel loop (loop diameter of 2.5 mm, wire
diameter 0.3 mm) is dipped into a pick-up solution. Retracting the wire loop quickly
out of the pick-up solution, in a direction perpendicular to the plane of the loop, will
result in a large drop. Slow withdrawal, sideways, will decrease the drop size. The
size of the drop is important since a larger drop will take longer to freeze. We have
currently two different pick-up solutions in our laboratory.
The first solution we started with is 2.3 M sucrose in 0.1M PB. The wire loop filled
with a sucrose droplet is introduced into the cryochamber. While looking through the
binocular, the droplet is brought to a position where you can see it in the binoculars,
yet as far as possible away from the sections. Usually some smoke (freezing water
vapor) is emerging from the droplet. Once it stops smoking press the droplet gently
on top of the gathered sections with the center of the droplet. Do not try to pick up
too many sections otherwise the surface of the droplet becomes overcrowded.
Looking through the droplet the sections are visible, and sometimes it is possible to
see the sections (over)stretch, soon after that the sucrose solution is frozen. Remove
the droplet from the cryochamber and allow the sucrose drop with sections to thaw.
Some people help the thawing by gently breathing on the drop. The effect of that is
questionable and when uranyl or fixatives are added to the retrieval solution (see
below) it may be a risky habit. After thawing, press the droplet with the section side
down on a Formvar carbon-coated grid. To check the quality of the sections, the
sucrose is removed by floating one grid on distilled water. After approximately 5
minutes the grid is removed from the water, air dried and viewed in the electron
microscope. If the sections are satisfactory, the other grids can be stored as
described in section Storage of thawed cryosections.
The second solution we use is a 1:1 mixture of 2.3 M sucrose in PB and 2% methyl
cellulose in distilled water which is prepared just before use. Prepare this mixture
fresh from cold solutions (4 C) and mix the constituents very gently (or use a
rotator) and keep it on ice during sectioning. Mixing at room temperature will give
white deposits, which is probably due to withdrawal of water from the methyl
cellulose solution by the sucrose before the mixing is completed. We started using
this second pick-up solution in 1996 after we found a vastly improved ultrastructure
in the sections picked up this way (Liou, Geuze et al. 1996). Since then this is the
21

standard way to pick up sections in our lab. The viscosity of this solution is much
lower and it freezes much quicker. The freezing is obviously crystalline. It is easy to
follow under the binoculars how it starts freezing at the edge (white rim) in the
corner of the loop. This is the moment to pick up he section. Wait longer and the
drop is completely frozen and the sections cannot be retrieved. With the MC/UA
solution the sections do not stretch on the droplet during pickup or thawing, so what
you see is what you get!! This implies that only the shiny flat sections give optimal
results. It needs some experience to use this pickup method successfully, but it pays
off at the end. The fine structure is much better as compared to sections retrieved
with 2.3 M sucrose.
In most cases 2.3 M sucrose or sucrose/methylcellulose are suitable pick-up
solutions, but 2-4% polyvinyl alcohol can be used also. When delicate lipid rich
structures are involved these pick-up solutions are not sufficient to retain lipids in a
cryosection. An additional on-section fixation is necessary to preserve lipid-rich
structures and the lipids in membranes. For these purposes Willisa Liou used a
mixture with final concentrations of 2% methyl cellulose and 2% uranyl acetate in
distilled water (Liou, Geuze et al. 1997)). The sections must be of outstanding
quality , thin (50 nm) and flat. She also had some success by thawing cryosection of
fresh, cryo-immobilized material in pick-up solutions containing mixtures of uranyl
and aldehyde fixatives. In other words she did the complete chemical fixation on the
sections (Liou, Geuze et al. 1996).

7.1.
Mounting of LM-sections
When the semi-thin 200nm thick sections are used for immuno-fluorescence (IF)
studies it is also an advantage when the sides have been trimmed, however these
section are rather ridged in comparison to ultrathin sections and it is not absolutely
necessary. 2.3M sucrose is a good pickup solution, the sections stretch slightly,
which enhances the accessibility of antigenic determinants. The z resolution of a
semi-thin section is 2-3 times better than an optical confocal section (in theory
0.2m but I reality 0.5-0.7 m). For IF the sections are placed on 3aminopropyltriethoxysilan coated microscope slides. With a diamond pen we scratch
lines of ~1.5 cm perpendicular to the length axis of the slide. One slide can carry 3
scratches at ~2cm distance from each other. After the scratching, make sure that all
glass splinters are removed with a paint brush or compressed air. 3 or 4 pick-up
drops (either 2.3 M sucrose or 1.15 m sucrose/1% MC) with semi-thin sections can
be placed alongside a scratch (scratch and sections at the same side of the slide).
Two different specimens needing identical immuno-staining can be placed on each
side of the scratch. For different antibodies, sections have to be placed along
different scratches. A fine wax pen is used to demarcate the area with sections
around a scratch. The wax line prevents the incubation drops from pulling away from
the scratch area. The wax should be allowed to dry at least a day. The sections with
sucrose can be stored in a cool place for several days prior to immuno-labeling.

22

7.2.
Storage of thawed cryosections
After the sections have been put on top of the grid, they can be stored in two ways.
When the grids are used within the next 24 hrs they can be transferred to 2% solid
gelatin plates (sections facing the gelatin). To prevent drying out the 3 cm plates are
stored in Petri dishes at 4 C in a closed container. Keep the gelatin cool during
sectioning. Second we introduced the storage of sections while still covered by the
pick-up drop of sucrose/methyl cellulose (Griffith and Posthuma 2002). The grids
remain attached to the stickered microscope slide on which they are prepared. In
that position the grids with sections are stored in a closed microscope slide storage
box at 4 C. It was found that drying under these conditions did not affect the
ultrastructure or immuno-reaction. For many molecules this is a perfect storage
method; however one should always test your antibody on a stored and freshly cut
section. Small or difficult to fix molecules may diffuse out of the section during this
storage.

8.

Immuno-labeling.

Figure 8.2: Immunolabeling with

IgG Gold

Figure 8.1: Immunolabeling with

ProteinA-Gold

As soon as biological research in the 1940s became aware of the fact that an
antibody (AB) can be used to mark the molecules to which they were directed (hence
called antigens) they became an indispensable tool in science. In theory it is a simple
procedure: First an antigen is purified, next it is injected into an animal of a different
species and nature does its job. The animal will recognize the antigen as a foreign
molecule and will produce antibodies which will recognize their antigen out of
millions of other molecules. Another widely used approach is genetic labeling. In this
case the protein of interest is tagged genetically with a relative small protein like
hemaglutinin, green fluorescent protein or with the smallest tag available at the

23

moment: tetracystein(Gaietta, Giepmans et al. 2006). The latter would be in theory

the most suitable tag, but there are as yet no antibodies available and Reash
reagents in combination with DAB cytochemistry must be used for detection in the
electron microscope. When ABs are available, they can be used to visualize
antigens on gels, blots, tissues, cells and (ultra-thin) sections with unprecedented
accuracy provided they are coupled to a suitable marker. To be able to see the
antibodies in the electron microscope an electron dense marker is necessary. At first
antibodies were coupled to enzymes that could produce an electron dense precipitate
(after osmium tetroxide fixation). Although extremely valuable, this technique has its
limitations: the reaction products are diffusible, sometimes difficult to discern from
the surrounding tissue elements and difficult to quantify. Visualization of more than
one antigen in a specimen is not possible. For immuno-electron microscopy a very
small discrete electron dense marker would be ideal. Faulk and Taylor (Faulk and
Taylor 1971) introduced the gold particle as a marker for the electron microscope.
Gold particles can be made in different sizes (Slot and Geuze 1981) and can be
coupled to a wide variety of biological active molecules : enzymes, antibodies,
toxins, lectins, protein A and many more. Since that time immunogold labeling is the
most widely used technique to visualize molecules in the (transmission or scanning)
electron microscope. For obvious practical reasons the primary antibody is not
coupled to an electron dense marker (availability of antibodies, shelf life). The
primary antibody is visualized by means of Protein A coupled to gold (Figure 8.1) or
an IgG (Figure 8.2).

8.1.
How many gold particles can be expected?
Before an immuno-labeling study is performed, one should always realize that there
should be enough molecules present to be visualized. In a thin section only a very
small part of the cell is visible. A 50 nm thick section of a standard 10x10x10 m
cell only represents 0.5% of the total volume or 0.3% of the plasma membrane!
Furthermore molecules will change during their lifetime in a specimen: they are
trimmed, glycosylated, phosphorylated, polymerization takes place, etc. Moreover,
the antigenic determinants can be destroyed by fixation and may not be accessible
to antibodies in the specimen thus further reducing the number of available
molecules. The highest reported labeling efficiency (LE) is approximately
10%(Griffiths and Hoppeler 1986), but often it is less than 1 %. When this standard
cell has 30,000 molecules on its plasma membrane, in the electron microscope
between 0.9 (LE=0.01) and 9 (LE=0.1) gold particles can be observed along the
membrane.

8.2.
Some background information
Molecules bind to each other, either very weak (no binding or even repulsion) or very
tight, the quality of this binding is described by a dissociation constant or Kd. A Kd <
10-8 describes a strong bond between AB and antigen whereas a Kd > 10-3 means that
the two are not tightly bound to each other. In the Bjerrum Plot in Figure 8.3
different theoretical plots are displayed. The curve with the solid circles represents a
genuine AB-antigen binding where, at a concentration of 5x10-8 mg/ml, 95% of all

24

the available antigen molecules are bound to an AB. The other curves represent the
binding of that same AB to other molecules. At a concentration of 10-4 mg/ml every
available antigen molecule is bound to an AB, but at the same time the AB binds to
other molecules as well due to
electrostatic or hydrophobic
interactions: background. The
solution to the background
problem is to dilute the AB to
the appropriate concentration
and block possible nonspecific
binding sites with molecules
that do not bind to the specific
AB but do bind to molecules in
the specimen. A blocking
agent should be selected
depending on the nature of the
Figure 8.3: Bjerrum plot of antibody-antigen binding.
Small Kd :strong binding, larger Kd weaker binding

nonspecific binding.

When antigens are very

abundant it is often quit
obvious whether the correct AB concentration is used. However when that is not the
case, statistics will give a clear answer whether the labeling is specific or not as
recently described in a brief yet comprehensive tutorial by Mayhew. (Mayhew 2005)

8.3.

Immuno-labeling strategies
In general, 3 different strategies can be
discerned: whole mount immuno-EM, Preembedding labeling and post-embedding
labeling.

Figure 8.4: Whole mount

immunolabeling.EM Picture of an
Enterococcus adhered to a FormvarCarbon coated grid. After fixation a
surface antigen has been labeled with
primary antibody and protein A-Gold.
Final staining with MC/UA. Bar=200 nm

Whole mount immuno-EM is in fact the

fastest method. Specimens are adhered to
grids or cultured on grids, (mildly fixed)
and labeled. When the labeling is performed
without any detergents present in the
incubation media it is most likely that only
the outside of a specimen will be labeled. In
this procedure the entire cell is present on
the grid and later in the electron
microscope. Therefore only rather thin,
non-electron dense, specimens can be used
in 60-120 KV electron microscopes. In
Figure 8.4 an Enterococcus has been
immuno-labeled for a surface antigen. The
same technique can be used to visualize
antigens on viruses, blood platelets and the
rims of flat cells. Immuno-labeling of the

25

interior of a specimen is possible when the specimens are treated with detergents
like saponin or Triton X100. This will generate small pores in the surrounding
membranes and (part of) the cytoplasm
and making the antigens accessible.(van
Dam and Stoorvogel 2002)
Closely related to the previous technique is
the pre-embedding technique. The
specimens are immuno labeled before they
are embedded in a resin and sectioned.
This is a very useful technique when the
distribution of surface antigens is studied.
To access the interior of a specimen (cell)
the plasma membrane has to be opened
prior to the labeling procedure. This can be
achieved by detergents or repeated freezethawing, which are rather mild treatments.
Cells can also be opened with an osmotic
shock or by mechanical forces like scraping
them from their support. Even after such a
harsh treatment, many organelles are still
intact but the cytoplasm is no longer
present. The absence of cytoplasm during
labeling increases the availability of antigenic determinants of cytoplasmic tails from
membrane associated molecules. It has
Figure 8.5: Pre-embedding labeling. Cells
were permiabilized and immunolabeled
been used for instance to visualize
for AP1and protein A-gold. Thereafter the
molecules on synaptic vesicles (De Camilli,
cells were embedded in EPON sectioned
Harris et al. 1983). In Figure 8.5 one of the
and stained with UA and lead. Arrow:
coat proteins (AP1) present at the Trans
clathrin coat, bar=200 nm
Golgi Network of HepG2 cells is labeled
with 5 nm gold particles. In this particular

Figure 8.6 : Immuno-double labeling of a Hela cell,

which was transfected with lung surfactant protein C
(SPC). Labeling: anti SPC > protein A-gold 10 nm and
anti PDI (resident ER protein, mouse antibody) >rabbit
anti mouse> protein A-gold 5 nm. Bar =200 nm.

26

case the cells were fixed wit

1.0% formaldehyde for 5 min
and scraped from the Petri dish
and embedded in 1% agarose.
Small blocks were incubated
overnight consecutively with
antibodies, gold probes and
afterwards embedded in EPON.
The last approach is postembedding immuno-labeling.
After the specimens have been
supported by a resin,
methacrylate or ice, the
sections are cut and the
labeling is performed on the
section. With a thickness of
70 nm most organelles will be
cut open and their interior is
exposed to antibodies. It is
obvious that the way in which

the specimens were treated will influence the labeling efficiency. Not only the fixation
will influence the number of available antigenic determinants, but also the chemicals
in the embedding procedure will destroy or mask epitopes. The immune reaction
takes place at the very surface of the section and the availability of epitopes is quite
different for each embedding method. Sections from specimens embedded in an
epoxy resin have a rather smooth surface which means that there are not many
epitopes available. The surface of hydrophobic methacrylates (Lowicryl HM20, LR
gold) resembles that of the epoxy resins, the more hydrophilic methacrylates
(Lowicryl K4M, LR white) have a rougher surface. Thawed frozen sections usually will
give the best LE because the antigenic determinants have not been exposed to
organic solvents and embedding media. Moreover their surface is rough and
antibodies can penetrate into the section (depending on the matrix density of the
organelle). Unfortunately gold particles ranging from 5 to 20 nm hardly penetrate
into a section due to their size and charge. (Stierhof and Schwarz 1989) This
problem can be tackled(partially) by using ultrasmall gold particles ranging from 0.8
to 2 nm. Due to their small size, these particles are hardly visible in the electron
microscope and need to be increased in size by means of silver or gold enhancement
(Yi, Leunissen et al. 2001).
8.4.
Immuno-double labeling
Due to the particulate nature and the well-defined sizes of gold markers it is possible
to label more than 1 epitope on a specimen.(Figure 8.6 and Figure 8.7) In theory
the number of different epitopes that can be labelled is only limited by available gold
particles. However, with an increasing number of immune-oreagents the greater the
chance of background labeling will be. In practice double labeling is frequently used
and occasionally also a triple labeling.
8.5.
The labeling procedure
During the immuno-labeling procedure the sections are incubated over a series of
different drops by floating the grids on top of the drops, sections facing the drop. For
most rinsing steps drops of 50-100 l are used which can carry up to 3 grids at once.
For antibody and immuno-gold solutions we use 5-10 l for each grid. The drops are
placed on a clean and flat surface. This is easily achieved by using a Parafilm sheet
that is adhered to a glass plate by some distilled water. The cover sheet of the
Parafilm is removed step by step while the incubation proceeds, so that the surface
is kept clean. The grids can be transferred by wire loops or by forceps. Using the
latter, less of the incubation fluid is transferred to the next drop especially when non
capillary tweezers are used, which renders the washing steps more efficient. The
adhering fluid may decrease the concentration of the immuno-reagent which are
usually in 5 l drops. To transfer a minimum of excess fluid (approximately 0.5 l),
pull the grids sideways and gently from the drops. Excess adhering fluid can be
removed with filter paper, but be careful: never allow the section side of the
grid to dry. Drying will damage the ultrastructure and a dry section surface tends to
be sticky to all kinds of proteins including immune-reagents. This will result in high
background labeling. The backside of the grid should stay dry throughout the
procedure. If a grid accidentally sinks and one wants to save it, wash it in distilled
water. Then dry the back side by carefully (but quick) wiping with filter paper and let
it float on clean distilled water before entering the incubation procedure again.

27

For a typical immuno-labeling of the sections, transfer the grids over the incubation
media mentioned successively. Routinely all media are buffered by PBS until the
washing steps in distilled water. Other buffer systems can be used as well. One
should be aware of the fact that uranyl will give precipitates with buffers that contain
much sodium. Copper grids should be replaced by nickel or even golden grids when
very long incubation procedures are followed or aggressive chemicals like in in situ
hybridization buffers are used. The incubations are at ambient temperature, unless
indicated differently. A detailed protocol is given in the protocol section.

8.6.
Background reduction
Both the support film and the sections will bind immuno-reagents due to
hydrophobic, charge mediated etc interactions. This problem can usually be solved
by covering these binding places with proteins. In the immuno-labeling for Tokuyasu
sections the sections are placed on 2% solid gelatin in a 3 cm petri dish. The petri
dish (with lid) is placed in 40C stove and kept there for 20 min. The melted gelatin
will cover many protein binding places and at the same time dissolve the gelatin in
between cells and pickup solution. After rinsing away the excess of gelatin, a
smaller, differently charged molecule like BSA is used to reduce the background even
more. There is a wide variety of blocking solutions: cold water fish skin gelatin,
ovalbumin, 1-5% (fetal) calf serum, dissolved fat free skimmed milk powder, diluted
goat serum, or 0.1-1% acetylated BSA (Aurion). The physical properties of the
antibody and section determine whether background reduction is successful and it
worthwhile to try different blocking agents or a combination thereof.
A different source of background might be free aldehyde groups, which I theory can
also bind immuno-reagents. Those are incapacitated with an amino acid like glycine,
but also Tris will do the job. Sometimes we use 0.1% -1% NaBH4. Sodium
borohydrate is very unstable (Tokuyasu, 1997) and it should be used immediately
after being prepared, or even refreshed once during an incubation period which is
usually 5 min. The hydrogen radicals will quench free aldehyde groups, reduce
double bonds in molecules induced by (GA) fixation and thus may restore
antigenicity. Additionally the tiny hydrogen bubbles may render the sections more
open which results in an improved penetration of the immuno-reagents during the
incubations. In case of immuno-fluorescence NaBH4 treatment takes away autofluorescence induced by GA.

28

8.7.
The immunoreaction
Immunoreagents are usually diluted in 1% BSA/PBS, but other blocking proteins or
buffers can be used as well. The dilution has to be worked out for each particular
antibody. The grids are floated on 5 l drops. They dry out easily, in particular when
long incubation times are used, so the drops should be kept in a humidified
atmosphere (wet tissue under a square Petri dish). In general, the duration of the
immuno-incubation is not very critical when high affinity antibodies are used. The
minimum time we use is 20 min at room temperature. The necessary time might be
longer when the concentration of antibodies is low, the temperature is low or the Kd
is high. Furthermore one should realize that antigens might be extracted from the
thawed sections during incubation. That may happen to molecules that do not react
with aldehydes, like membrane lipids, but also soluble proteins may escape easily
when the fixation is weak (Posthuma, Slot et al. 1987). In such cases it is better to
keep the incubation short. On the other hand, proteins that are integrated in
membranes or bound to the cyto-skeleton will not easily escape and labeling of these
may be favored by longer incubation periods, during which soluble compounds may
be extracted, so that penetration into the section may improve. When the incubation
is extended to the next day, we usually keep the grids overnight at 4 C in a
moisture atmosphere. In that case the antibody should be further diluted (~10
times) to prevent that non-specific binding becomes a problem.
When the final marker does not react with the primary IgG it is necessary to use a
bridging step. For example goat IgGs or many mouse monoclonal antibodies will
not react with protein A. Be aware of the fact that background may increase with
each step and that the distance between epitope and gold particle increases after
each step. There is one major advantage of using one (or more) bridging steps:
more epitopes are visualized because the AB can penetrate into a some parts of a
section thus increasing the labeling efficiency. (Slot, Posthuma et al. 1989).

8.8.
Controls
To check for non-specific labeling by the primary antibody, the bridging antibody, or
the PAG or IgG probes, sections should be incubated in parallel and treated with an
identical incubation procedure as the experimental grids
Checking for non-specific labeling induced by the primary antibody can best be
performed by processing and labeling tissue or cells that are similar to the specimen,
but are lacking the antigen. This is a feasible control in all studies on transfected
gene products or when knock outs are available. The second best control is to
perform a labeling with the preimmune serum. This should not give any gold
particles when a similar dilution is used. It is important to supplement these tests
with electrophoresis and immuno-blotting. Unfortunately the results do not always
match perfectly: antigen molecules in solution may have different binding
characteristics than molecules in sections. Another test would be to mix AB and
antigen prior to the incubation. We often found huge clusters on our sections after
incubation pre-absorbed AB solutions.

29

8.9.
Rinsing
After each immuno-reagent the grids have to be rinsed. The grid is removed from
the droplet sideways with tweezers and usually only 0.5 l is left. Quickly transfer
the grid to the next (rinsing) drop of approximately 75 l. 3 rinses will in theory
dilute the antibody 150 x 150 x 150 = 3,375,000 times which is usually more then
sufficient to abolish any remaining activity.
8.10.
The marker
The best visible markers for electron microscopy are gold particles bound to IgGs ,
protein A, lectins etc. They are manufactured in a wide range of sizes (0.8 40 nm)
and allow double, triple or even quadruple labeling. Of course all of these markers
can bind nonspecifically and should always be diluted to the proper concentration in
a background reducing solution. We prefer Protein A-gold over any other molecule
since the protein A can be easily purified, gives very reproducible results and is more
precise since only one PAG particle can bind to 1 Fc part of a primary or bridging
antibody. Furthermore it allows double labeling when the primary antibodies are
from the same species, because the Fc biding site for protein A is destroyed by a
brief glutaraldehyde fixation. However PAG has also some disadvantages. It does not
bind to the all the IgG subclasses of every species. Due to its relative small size
(Mwt 42 kD) as compared to IgG (Mwt 176 kD) it may not recognize IgGs just
beneath the section surface since the charge of the gold usually prevents the gold
particle to enter the section.

8.11.
Stabilization of the immune reaction
When primary antibody, bridging antibody and marker are in place, it is useful to
stabilize this reaction with a fixation step. The laws of physics tell you that although
firmly bound, each of the immuno reagents can (and will be) detached in time. To
prevent that a 1% glutaraldehyde fixation is used. This is particularly useful in an
immuno-double labeling. Besides stabilizing the immune reaction it also destroys the
protein A binding site on the Fc portion of the IgG, so it will not be recognized in a
double labeling procedure by the second PAG particle. The GA treatment can be a
problem when the second immuno-reaction is GA sensitive. Fortunately we noticed
that this on-section GA fixation is often not as bad as one would expect for antigenic
sites that are killed when GA is used for the initial cell. There are two possible
explanations for that. First, GA sensitivity is often a matter of penetration barriers
that are created by the cross-linking effect of GA. It may well be that these barriers
cannot be formed anymore in a weakly fixed section that has gone through a series
of incubations. During these incubations many molecules have been extracted, that
could be part of these barriers. Second, if GA cannot be used for initial fixation, one
usually fixes with FA. Since both aldehydes react primarily with the same groups,
largely amines of protein molecules, it may well be that the GA reaction is rather
mild with proteins in sections that are prefixed with FA.
When GA fixation abolishes the labeling FA or free protein A can be used.

30

8.12.
Double labeling procedure
A double labeling procedure is essentially(Figure 8.7) two consecutive single labeling
procedures. After stabilization of the first immune reaction possible free aldehyde
groups have to be quenched, with 20mM glycine/PBS. Next the single labeling
procedure is performed once more with a different size PAG. As mentioned it is
important that bridging antibodies used for this stage of the double labeling
procedure are not reactive to the IgG of species used in the first labeling. Therefore
it is often not possible to use 2 monoclonal antibodies in a double labeling procedure
without precautions. Only mouse IgGs of type 2a, which can be labeled directly with
PAG without using a bridging antibody are suitable for double labeling in this
sequential procedure.
Even within the limitations set by
obvious cross reactions between
the 2 antibodies one occasionally
encounters less understood
interactions between the first and
second labeling. Sometimes the
second gold seems to stick to the
first, even without bridging
antibody. A possible explanation
is that even after the GA
treatment, protein A still has
some affinity to particular
subclasses of immuno-globulins.
We also encountered cases where
the first gold sticks to some
extent to the second antigen. This
can only be explained when (part
of) the first immuno-complex
dissociates from its initial site and
binds
again at some stage during
Figure 8.7: Immuno-double labeling with protein Athe second labeling procedure via
gold in different sizes and two primary IgGs
sensitive to protein A. This is possible because
the second specific antibody. It is
binding site for protein A is destroyed with with
a not an uncommon phenomenon
glutaraldehyde.
that the labeling intensity for one
or both of the antigens is lower in the double labeling than in single labeled sections.
Specific characteristics of each different antibody preparation make that these
problems are not always as manifest. To deal with them, it is important to reverse
sequences of labeling for the two antigens and prepare single-labeled sections when
you evaluate double-labeling patterns.
An alternative for the sequential procedure is based on IgG labeling and can be
applied when both specific antibodies are generated in 2 different species. For
instance: anti-A from mouse and anti-B from rabbit. They are labeled by 2 different
IgG probes: goat anti-mouse IgG-Gold 5 nm and goat anti-rabbit IgG-Gold 10 nm
respectively. This double labeling can be performed conveniently in one labeling
procedure: In the primary antibody step the grids are incubated in a mixture of antiA and anti-B and next one uses a mixture of the 5 and 10 nm IgG probes.
31

Las but not least interactions of Immunoreagents can be abolished when the labeling
is performed on different sides of the section. This is virtually impossible in Tokuyasu
sections, but can be used with resin sections.

8.13.

Section Light Electron Microscopy (SLEM) labeling.

When rare events have to be
observed in the electron
microscope the searching time
can be very long, whereas this
rare event can be easily found
in a light microscope. In the
group of Tacchetti (Cortese et
al 2009) a very useful labeling
procedure was developed. that
can be of great help in this
respect. The sections are
mounted on locater grids and
labeled with a primary
antibody. The next step is a
fluorescent secondary antibody
followed by protein A-gold or
IgG-gold. The fluorescent
marker can be found back in a
sensitive light microscope, and
retraced on the basis of LM
micrographs in the electron
microscope. (Figure 8.8)

Figure 8.8: Slem labeling with specific primary

antibody followed by a fluorescent antibody and by
Protein A

8.14.
Immuno-fluorescence labeling
Semi-thin sections on glass-slides are labeled for IF by essentially the same
sequence of incubations as described for EM, except that PAG is replaced by a
fluorochrome coupled to a secondary IgG. When the specimen is fixed with GA, the
sections are treated with 0.1% NaBH4 (1 mg/ml) in PBS for ~10 min. prior to the
labeling procedure to reduce autofluorescence
For IF staining we cover the wax delineated areas with incubation medium in the
labeling steps ; ~30 l should be enough. The washes in between and after can be
intensified by gentle flushing with a Pasteur pipette. Be careful not to flush
antibodies over neighboring areas with sections that are labeled for different
antigens.
After the final wash with distilled water, make the surface nearly dry, place tiny

32

drops of Vectashield or Prolong Gold etc. in each area with sections and place a
coverslip over the sections. Carefully avoid air bubbles and too embedding medium.
Let it spread out for an hour or so, remove excess of medium and then seal the rims
with nail polish. The slides can be stored in the dark at 4 C for months.

9.

Contrast and support

Without the use of heavy metals like uranium, the section is barely visible in the
electron microscope. Therefore the constituents of the ultrathin section are
decorated with heavy metals. After being thoroughly rinsed (any salt remnants will
give unwanted precipitations) the grids are first stained with 2% uranyl
oxalate/acetate, pH 7 for 5-10 min. The purpose of this step is not very clear. Some
say it enhances contrast but in case of doubt one might consider to omit it from the
procedure. Tokuyasu originally introduced it to stabilize membrane lipids (Tokuyasu
1976). Next the excess of Ua-oxallate is removed by rinsing briefly with water and
next transferred to a solution of Methylcellulose (1,8 %) Uranylacetate pH 4 (0.4%)
on ice. After 2 short rinses with this solution it is left there for 5-10 min. All MC/UA
drops should be on ice. MC solutions are less viscous at lower temperature, so that
they can penetrate better into the sections. Cover the drops with a Petri dish to
prevent drying. Do not leave them too long on the ice-cold MC/UA when the humidity
is high, condensation of water might occur and ruin the grid.

Figure 9.1: Final embedding with MC/UA. A: grid has just been picked up from a cold Mc/UA
solution. B: the excess of MC/UA is removed from the grid. C: a thin layer of MC/UA is left
(upper) and dried at room temp (lower). D:When not enough MC/UA is left, the section has
too much MC/UA on top and the section will hardly be visible in the EM.

Instead of MC a mixture of 2-4% polyvinylalcohol (Mwt 10,000) and 0.4% UA can

be used at room temperature. The grids can be removed from the MC/Ua solution by
means of a wire loop with an inner diameter between 3.5 and 4 mm. Push the loop
into the MC/UA drop at some distance from the grid, move it under the grid and lift

33

the grid from the MC/UA drop. Now the grid is in the center of the loop with excess
MC/UA hanging underneath. Tilt the loop and grid to an angle of 45-60 (the excess
MC/UA downwards) and touch the filter paper with the loop. The kind of filter paper
and the number of layers determines the speed with which the MC/UA is drained
from the grid. Two layers of Whatman no 50 on a piece of Parafilm is a good start.
The amount of MC/UA on the grid should be enough to make contact and disappear
into the filter paper, leaving behind an even thin film on the surface of the grid,
which remains in the loop center. As soon as the MC/UA starts to disappear into the
filter paper move the grid gently sideways until the removal of MC/UA stops. The
smaller the angle and the slower you move sideways the thinner the final film will
be. When the grids are drying, the concentration of UA will increase and the UA
precipitates to the nearest molecule, thus generating the beautiful positive-negative
contrast we know from standard Tokuyasu sections. After drying at room
temperature for at least 30 min the section is visible as a bluish islet in a golden sea.
They can be removed from the loop with fine tweezers. Be careful with the uranium
in this part of the procedure. Discard the pieces of absorbing filter paper and
Parafilm properly. Tokuyasu sections are very stable in the electron microscope and
can be kept in a grid box for years.

10.

Carbon-Formvar coated grids

We use carbon-coated Formvar films that are spread over hexagonal copper grids.
Nickel grids can be used as well and have the advantage of being more inert to
chemicals, like PB or PBS, but their magnetism can be a nuisance. When the
Formvar film is without holes the interaction between buffers and the copper grid can
adequately be avoided. Only in case of long storage on PB or PBS this may be a
problem. Sometimes the Formvar is damaged along the grid-bar edges. Then
incubation media can penetrate to the backside of the grid, dry out and cause typical
precipitation zones along the bars. It has been found that this phenomenon occurs in
particular with certain brands of grids. Formvar as such is a good support for resin
sections; however for thawed (and frozen) sections the film should be covered with a
thin layer of carbon which makes the section stick to the film and reduces the
charging of a grid in the electron microscope. The carbon is extremely pure and
essentially anything will adhere to it. Therefore carbon coated grids can only be
stored for a limited amount of time (3 months max).
The Formvar film on the grids is coated with carbon to render it hydrophilic and
better electrostatic properties. At 2x10-5 mbar a very thin layer of carbon is
evaporated onto the film using standard techniques. A good indication of the
thickness can be obtained by placing a small piece of adhesive tape on the sticker
with grids, which is removed after carbon evaporation. The border line between non
coated and carbon-coated surface should be just visible.

34

11.

Protocols

In this section you will find step by step protocols as we use them in the Cell
Microscopy Center. This does not mean that every protocol has to be treated as the
Holy Grail, use them as guidelines to develop your own protocols. Often protocols
lack information regarding little tricks, that are often necessary to generate a good
result. We have tried to cope with these items by introducing the tips and tricks
footnotes (for instance 1), that refer to a useful comment regarding that particular
step. In [square parenthesis] the brand and catalogue number of the used chemicals
are mentioned when this is important. When solutions like PBS or carbon Formvar
coated grids are mentioned in a protocol, you can find the recipe in the recipe
section or in an other protocol.

Read footnotes carefully, it may save you some time.

35

11.1.

Diamond knife cleaning

Materials and equipment

1.
2.
3.
4.
5.
6.
7.
8.
9.

Binoculars
Diatome polystyrene rod, beveled to an angle of 90
Firm Balsawood rod (see remarks) beveled to an angle of 90
Glass Petri dish (10 cm diameter)
Glass Petri dish (depth 4.5 cm) filled with distilled water
Gloves
Razorblade cleaned with acetone
Squeeze bottle with distilled water
Squeeze bottle with ethanol 99.5%

Reagents setup
1.
2% Decon 90 in distilled water in Petri dish
2.
2% Decon 90 in distilled water, freshly made every week
3.
Glass Petri dish (depth 4.5 cm) filled with distilled water
4.
Latex gloves
5.
polystyrene strips beveled to an angle of 90
6.
razorblade cleaned with acetone
1.
Procedure
Before and/or after cutting:
7.
Put gloves on
8.
Rinse the knife under running tap water for 30 sec. 1, 2
Figure 11.1: rinsing with tap
water

9.
Rinse knife with distilled water
10. Rinse the
11. Take the deep Petri dish with distilled water (under the binocular)
Hold the knife under the water surface and clean it with the beveled polystyrene rod
by gently running the rod across the cutting edge 6-8 times. Use each time a fresh
place on the rod. 3, 4, 5

1
2
3

4
5

Do not use calcium rich tap water (use distilled water in this case)
Rinse always in the direction from the cutting edge down to the bottom of the knife.
During cleaning with the rod (polystyrene or Balsawood), be sure your knife and rod are
wet. If the rod is too dry you will contaminate the knife and this will cause a lot of
compression
Instead of polystyrene you can use Balsawood
Dry the knife always immediately with a photographers blower after rinsing with ethanol.
Only until the knife-edge is dry. Remnants of evaporated ethanol on the knife-edge are
deadly for sectioning, no thin sections can be obtained.

36

Figure 11.2 :Left panel : Cleaning diamond knife under binoculars in a petri dish with
water. Middle panel: Cleaning diamond with styrofoam: just enough pressure to get 1-2
mm deep impressions. Right panel: Above: rinse with alcohol, below : blow dry with a
photographers blower

12.
13.
14.
15.
16.

Repeat if necessary
Rinse the knife under running tap water
Rinse the knife with distilled water
Rinse with ethanol 99.5%
Dry the knife-edge directly with a photographers blower. Blow the droplets
from the knife-edge down to the bottom
17. Be sure the knife is really dry before you put it in the cryochamber or put it in
the box.
New knife: Use the knife directly from the box and put it in the cryochamber
Cleaning with Balsawood rods
1.
2.
3.
4.
5.
6.
7.
8.

Boil Balsawood (10x10cm) for 10 min. in distilled water

to remove minerals Figure 11.3
Rinse with distilled water
Cut small strips parallel with the wood grain and leave
the strips of balsawood overnight in 2% Decon
Dry the strips and store them.
For daily use: take a rod from the storage, bevel it to
an angle of 90
Put the rod for a few minutes in the 2% Decon
Run it across the cutting edge. Avoid bringing the
knife-edge in contact with the dark grains in the
Balsawood as this can damage your knife.
Dry the knife always immediately with a photographers
blower after rinsing with ethanol till the knife-edge is
dry. Remnants of evaporated ethanol on the knifeedge are deadly for sectioning, no thin sections can
be obtained.

37

Figure 11.3:Boiling of
balsa wood

Protocol according to Helmut Gngi:

1.
2.
3.

Bevel a Diatome polystyrene rod to an angle of approx. 90.

Dip in ethanol 50% (in water)and shake off the excess.
Gently run the rod across the cutting edge without applying lateral pressure.

If sections or debris dry on the knife edge: Place the knife in distilled water
overnight and clean the knife as described above.
Cleaning after sectioning is always necessary, cleaning before sectioning (probably)
not!
Caution__________________________________________________
1.

2.

3.

Dry the knife always immediately with a photographers blower after rinsing
with ethanol. Only until the knife-edge is dry. Remnants of evaporated
ethanol on the knife-edge are deadly for sectioning, no thin sections
can be obtained.
Do not use DECON 90 overnight with trimming diamonds or standard diamond
knives. The aluminum is corroded by the DECON. It will lose its nice blue
color and the aluminum salts may change the sectioning properties of the
diamond.
Use only polystyrene rods from Diatome and bevel the rod to an angle of
approx. 90

38

11.2.

Fixation

Reagents
1.
2.
3.
4.
5.
6.

(para)formaldehyde (PFA): 16% FA prills, [Sigma 441244]

Acrolein: 90% wt/vol. [BDH 27044]
Glutaraldehyde (GA): 8% EM grade [Polyscience 00216]
PBS
PBS/glycine 0.15%
Phosphate buffer (PB):

Commonly used fixatives in immuno-EM

1.
2.

4% FA in 0.1 M PB, 2-96 hr.

2% FA + 0.2% GA in 0.1 M PB, 1-2 hr
Furthermore many other combinations have been used in the past like:

3.
4.
5.
6.
7.
8.

2% FA in 0.1 M PB, 2-96 hr.

2>4(>6>8)% FA (10 minutes each step) in 0.1 M PB,1-96 hr
1% Acrolein + 0.1% (or 0.5%) GA in 0.1 M PB, 1 hr.
2% FA + 1% Acrolein in 0.1 M PB, 1-48 hr.
2% FA + 0.5% GA in 0.1 M PB, 1-2 hr.
2% GA in 0.1 M PB, 2 hr.

Evidently PB can be replaced by 0.1M PIPES, HEPES or 0.1M PHEM buffer.

Procedure
Cell suspensions:
1.
2.
3.
4.
5.
6.
7.

Mix a volume of cells gently with same volume of double concentrated fixative 1
Fix the cells at room temperature for 5 min.
Let the cells sediment, if necessary gentle centrifuge (< 200 g, 5 min.) 2
Remove the supernatant
Resuspend the pellet in 1-1.5 ml fresh single concentration fixative
Proceed the fixation
After fixation let the cells sediment, if necessary gentle centrifuge (< 200 g, 5
min.)

Adherent cells:
1.
2.
3.
4.
5.

Cells are grown in at least a 6 cm Petri dish (confluent).

Refresh the culture medium (3 ml) fresh the day before fixation
Add 3 ml double concentrated fixative at culture temperature (normal
strength buffer)
Mix gently but quick
Fix the cells at room temperature for 5 min.

After some treatments fixation on ice is necessay. This is possible but the ultrastructure will
be less good.
2
In case of floating cells use, after centrifugation, the upper layer. Bring to a new eppendorf
vial and continue the procedure

39

6.
7.

Remove the fixative and refresh with single strength fixative

Proceed the fixation

Tissues:
Whole body perfusion via abdominal aorta
1.
2.
3.
4.
5.
6.
7.
8.
9.

Sedate the animal with a barbiturate (for example Nembutal, 60mg/kg body
weight)
Open the abdominal cavity
Insert a needle in the abdominal aorta
Make an incision in the inferior vena cava close to the liver
Start the perfusion pump
Flush the vascular bed with PBS (to which an anticoagulant can be added to
prevent clotting) to remove most of the blood
Flush the fixative for 5-10 min.
During the flush ~1.5 times the blood volume (10ml for a 200g rat) is passed
through the circulation in 0.5-1 min. Fixative follows with the same flow for
5-10 min.
Excise the needed tissues and proceed with an immersion fixation

Whole body perfusion via left ventricle perfusion

1.
2.
3.
4.
5.
6.
7.
8.

Sedate the animal with a barbiturate (for example Nembutal, 60 mg/kg body
weight)
Open the abdominal cavity
Cut the diaphragm (breathing will stop!!)
Tighten an arterial clamp at the tip of the sternum
Cut through the ribs laterally with scissors
Bend the sternum towards the head
Insert a needle into the left ventricle
Proceed with step 5 from the whole body perfusion via aorta.

In this procedure the breathing stops as soon as the chest is opened, which urges
the operator even more to handle the subsequent steps as quick as possible. On the
other hand inserting the needle in the ventricle is easier than in the aorta, especially
in small animals.
Yeast
1.
2.
3.
4.
5.
6.
7.

Transfer 10 ml of yeast culture (OD600 = approx. 1) directly from the

incubator to a 50 ml Falcon tube containing 10 ml of 2x concentrated
fixative (4% PFA + 0.4% GA in 0.1M PHEM buffer) at room temperature.
Incubate 20 min at room temperature on a roller bank.
Centrifuge at 1000 rpm for 7 minutes.
Decant supernatant and add 10ml of 1x fixative (2% PFA + 0.2% GA in 0.1M
PHEM buffer)
Incubate further for 2 hours at room temperature on a roller bank.
Centrifuge at 1000 rpm for 7 minuets.
Resuspend the pellet in 1 ml of 0.1M PHEM buffer and transfer to a 1.5 ml
microfuge tube

40

Storage
1.
2.

Remove the fixative

Add 1% FA in 0.1M PB, store at 4C

Shipping
1.
2.
3.
4.

Remove the fixative

Fill the flask completely with 1% FA and wrap in Parafilm.
Try to avoid petri dishes but..In case of a dish: fill the dish completely with
1% FA, put parafilm on top, close the lid, wrap again with Parafilm,
unfortunately this is often not sufficient to prevent leakage.
During long distance shipment by plane, the fixed sample may become frozen
and the ice crystals may cause substantial damage to the ultrastructure, in
particular when only formaldehyde has been used for fixation. Therefore it
is advisable to ship them in >1.8M sucrose, to which 1% FA can be added.

Do not store fixatives and antibodies in the same fridge, the fixatives fumes may affect the
AB reactivity!

41

11.3.

Carbon-Formvar coated grids

Materials and equipment

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.

Adapted funnel with two stop-cock valves in its outlet

Carbon coater
Chamois
Erlenmeyer 50 ml
Filter paper
Glass beaker
Grids
Lens paper
Microscope slides
Microscope slides with a white (preferably hydrophobic) sticker or Parafilm
Photographers blower
Razorblade
Vortex

Reagents
1.
2.
3.

Acetone
1% NH4OH
1.1 % Formvar [Formvar= Vinylec E, SPI 02492-RA ]in dry Chloroform.

Procedure
Cleaning of the grids:
1.
2.
3.
4.
5.
6.

Put the grids in a 50 ml Erlenmeyer 1

Add 25 ml acetone
Stir on a vortex 2
Discard the acetone
Repeat step 2-5 2x
Dry the grids on filter paper

Preparation of Formvar film

1.

2.

Clean a microscope slide:

Wipe with a chamois
Wipe with lens paper
Remove the dust with a photographers blower 3
Pour the Formvar solution gently in an adapted funnel:
Level of Formvar in the funnel is 2/3 of the length of the slide
Upper stop-cock valve is used for regulating the flow

Not too many grids at once, they will stick to each other and are not clean enough
(grids fall off the film) or dont dry enough (grids become rusty)
When the grids are dirty:
1. Rinse with distilled water
2. Replace the water with 25ml of 1% NH4OH2
3. Vortex until the solution becomes lightly blue
4. Discard the ammonia solution
5. Rinse 10x with distilled water to remove all traces of ammonia

When the microscope slide is too clean the film doesnt come off
42

2.
3.
4.
5.
6.
7.
8.
9.
10.

11.
12.

13.
14.

Lower stop-cock valve is used for switching on or off

Set the upper valve in such a way that the Formvar solution flows out in
10-15 sec.
Place the clean slide vertically in the center of the funnel, use tweezers
Cover the top of the funnel
Open the lower valve with one turn
Remove the slide from the funnel
Loosen the film at the edges of the slide:

Use a clean razorblade to cut 0.5 mm from the edges or

Wipe the edges with lens paper

Fill a beaker with clean distilled water
Clean the water surface by wiping it with a glass rod
Condense the slide by breathing heavily on it
Bring the slide slowly in vertical position into the water 1
The film will float on the water surface
The color of the film should be silver-white (thickness ~50 nm), use a
lamp
Place the clean and dry grids on the film, shiny side up, avoid irregularities in
the film
Press a microscope slide with sticker gently on the film with grids down into
the water 2
The film with grids will attach to the sticker
Press far enough into the water so that the film is totally submerged
Remove the slide with film and grids slowly from the water (avoid adhering
water)
Dry the slide with film and grids at room temperature overnight (dustfreee)

Figure 11.4: Preparation Formvar coated grids

1

When the film is too thick (yellow) decrease the flow speed (slower). When the film
is too thin (gray) increase the flow speed (faster)
When the microscope slide is too clean the film doesnt come off.
43

Checking the film quality:

1.
2.
3.

Put a dry grid in the electron microscope

1,2,3
Check for irregularities, holes and dirt at 20.000x
Test the strength by removing the objective aperture and focusing the beam.
The film should not break

Carbon coating of the Formvar film:

1.
Place a small piece of adhesive tape on the sticker with grids
Evaporate a thin layer of carbon at 0.01 Pa (10-4 Torr) onto the film Remove the
small piece of adhesive tape. The border line between carbon coated and non-coated
surface should be just visible 4,5

2
3

4
5

When the stickers sucks too much water parafilm can be used (wrapped around a
microscope slide) also in case of one slot grids this is preferable
Dirt/Irregularities: make a new Formvar solution, clean all the glassware meticulously.
Store the slides with Formvar films and grids, carbon coated in a dust free box (3-4
months)
Non carbon coated Formvar coated grids can be stored for at least 1 year

Too much carbon: brittle films; Too little carbon: antibodies stick to the film
44

11.4.

Correlative light electron microscopy (CLEM)

Materials, equipment and reagents

1.
2.
3.
4.
5.
6.
7.
8.
9.

See formvar films

See fixation
See embedding
See cutting
See labelling
Gridded coverslips
Punched petridishes
Dental wax
Copper grids 50 mesh

Procedure
Prepare culture dishes
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.

13.

Wash the gridded coverslips with water and ethanol, let them air dry.
Attach the gridded coverslip to a slide with a paper sticker.
Make a formvar film on a clean watersurface (see EM protocol)
As a alternative: attach the gridded coverslip with a little dental wax to the
microscope slide (grid to the top so the grid will be covered with the film)
and use that combination to cover the grid with the film.
Let the slide and coverslip dry at roomtemperature.
Place a drop of 1% gelatin on the Formvar-coated coverslip and incubate for
30' at RT.
Gently remove the 1% gelatin and rinse 2x with ddH2O.
Fix the gelatin immediately with 1% GA for 15' at RT. Do not let the gelatin
dry on the coverslip! This will lead to a thicker gelatin layer and thus
background in the fluorescence microscope.
Rinse the coverslip 2x with ddH2O and let it air dry
Place a punched petridish upside down on a metal cylinder on a heating plate
of 70-75C and place the coated coverslip upside down on the petridish.
This will prevent the dental wax from solidifying as soon as it touches the
dish/coverslip
Melt the dental wax with a soldering iron and place a drop next to the
coverslip. Capillary activity will distribute the wax between the coverslip
and petridish. This will leave the backside of the coverslip clean (and
level), which is important for flat embedding later.
Sterilize the home-made MatTek by placing them under the UV lamp for
minimal 30 min.

Transfection
1.
2.

The transfection protocol is dependent off the goal of the experiment. So it is

not explained in this protocol.
Make sure the cell density is quite low, too much cells makes mapping the cell
of interest later on very difficult.

45

Microscope
1.
2.
3.
4.

Load the cells with dextran568 if necessary (1:100, 2 hours 37C)

Wash the cells with MEM image medium
Mark and/or draw the approximate location of the cell you are going to image.
Image the cell of interest and fix just before the last frame with 4% PFA
0.05% GA, be careful opening the doors because this might cause the
cell to go out of focus.
5.
Leave the initial fix on the cells at least 2 hours or overnight.
6.
Replace the fixative with PBS and make a z-stack of the cell
7.
Map the location of the cell by making DIC images of the cell + grid using a
40x lens
8.
Stitch the picture together and note the location off the cell
9.
Post fix the cell in 2%PFA 0.2% GA overnight
10. Store the cell in 1% PFA until flat embedding

Flat embedding
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.

Rinse with PBS and PBS/Glycine

Detach the coverslip from the dish (keep the cells wet)
Mount the coverslip on an objectslide (cells under) with mowiol
Mark the correlative cell with a nail polish dot on the backside of the coverslip
and let dry for 15 min.
Lift the coverslip from the objectslide by dissolving the mowiol in PBS
Put the coverslip in a 3 cm petridish (cells up)
Rinse in PBS
Rinse in PBS / 0.1% BSA, remove and directly add 800 l 6% gelatine in PBS
37C.
Put the dish+coverslip 30-60 min. at 37C in wet chamber
Solidify at room temp 30 min.
Rinse with PBS / 0.1% BSA remove with filter paper and directly add 400 l
12% gelatin + erythrocytes 37C.
Solidify at 4C. 30 min.
Fix the cells and gelatin layers for 30 min. at 4C in 0.5% PFA in PBS
Rinse 2 x fast with cold PBS
Add 2.3M sucrose 4C. replace after 30 min. (fresh sucrose)
Infuse for 3 days at 4C.
Cut out a block of gelatin with the correlative cell (nailpolish mark)
Stain the block in 2.3M sucrose in 1% Toluidine blue 30 min.
Rinse in 2.3M sucrose
Mount the blocks on the pins
Freeze in LN2

Ultrathin sectioning
According the protocol in the manual. Only trim the sides, not the front of the block.
Directly start cutting ultrathin, cut serial sections.
Labelling
According the protocol in the manual.

46

11.5.

Gelatin embedding

Materials
1.
2.
3.
4.
5.
6.
7.
8.

37C stove
Double edge razor blade
Eppendorf tubes
Fume hood
Gloves
Rotor at 4C
Swing out rotor
Teflon coated razor blade

Reagents
1.
2.
3.
4.
5.

PBS
1% gelatin
12% gelatin
PBS/Glycine 0.15%
2.3 M sucrose

Additional for flat embedding:

1.
2.
3.

Rocker at 4C (cold room)

PBS/BSA 0.1%
12% gelatin + fixed erythrocytes (mix 5 ml 12% gel + 100 l ery-pellet)

Procedure
Cell suspension
1.
2.
3.
4.
5.
6.
7.
8.

Pellet the cells by mild centrifugation (< 200 g, 5 min.) 1

Remove the fixative
Rinse 3 x with PBS
Rinse 10 min. with PBS/Glycine
Suspend the pellet in 1 ml 12% gelatin 37C in a 1.5 ml Eppendorf tube for 10
min. at 37C
Prepare a cell pellet by centrifugation, preferentially with a 90 angle or swing
out rotor.2 The centrifugation results in a high density of cells in the final specimens. If
Solidify the gelatin cell pellet on ice (30 min)
Cut off the tip of the tube that contains the pellet with a stainless steel Teflon
coated razor blade. Cut the tip of the vial into two halves and put in a drop
of ice-cold PBS. After a 15-30 minutes the gelatin is separated from the
plastic of the vial.3

In case of floating cells use, after centrifugation, the upper layer. Bring to a new eppendorf
vial and continue the procedure
If the infusion or the centrifugation are too short the blocks become very soft and difficult to
cut
When the gelatin sticks to the plastic, place the specimen in 2.3 M sucrose (in buffer) for 30
min. The pellet will shrink and loosen from the plastic.

47

9.

Cut small blocks of desirable shape and size with a double edged razor blade
from the specimens. Non-fixed 12% gelatin cuts easier at 0-4C, so either
use ice or the cold room. Rectangular blocks with edges up to 0.5 mm are
fine for thin sectioning. 1
10. Infuse with 2.3M sucrose at 4C (4 hrs overnight) on a rotor (rotation speed
~120 rpm, angle 45-90)
Adherent cells
1.
2.
3.
4.
5.
6.

Follow step 1-4 from the cell suspension

Add 1.5 ml of 1% gelatin at room temperature
Scrape the cells with a cell scraper and collect them in a 1.5 ml Eppendorf
tube 2
Centrifuge
Replace the 1% gelatin by 12% gelatin 37C and infuse for 10 min. at 37C
Follow step 6-11 from the cell suspension

Tissue
1.
2.
3.
4.
5.
6.
7.

After fixation prepare small blocks (<1 mm3) or thin sheets (thickness ~1
mm) using a sharp razor blade
Rinse 3x PBS
Rinse 10 min. PBS/Glycine
Infiltrate with 12% gelatin at 37C for 15-30 min. while gently swirling the
specimens 3
Solidify the gelatin-tissue blocks at 4C
Cut off the excess of gelatin 4
Infuse with 2.3M sucrose at 4C (4 hrs overnight) on a rotor (rotation speed
~120 rpm, angle 45-90)

Cell monolayers on filters

1.
2.
3.
4.
5.
6.
7.
8.

Remove the fixative

Rinse 3x with PBS
Rinse 10 min. with PBS/Glycin
Remove the filters from their holder and cut into small square pieces (<1x1
mm).
Infiltrate the filter fragments in 12% gelatin in buffer at 37C during 15-30
min while gently swirling the specimens.
Solidify the gelatin-filter blocks at 4C
Cut off the excess of gelatin , the filters should still be surrounded completely
with gelatin.
Infuse with 2.3M sucrose at 4C (4 hrs overnight) on a rotor (rotation speed
~120 rpm, angle 45-90) 1

Be aware of the orientation of elongated cells or tissue in the block. Cut the block not in a
square shape but in a rectangular shape
Scraping in buffer is possible but the cells float. gelatin decreases the surface tension and
cells will sink and more easy to collect
For good penetration of the gelatin the infiltration is often done in three steps, in 2, 5 and
10% gelatin successively. Each step 10-30 min at 37 C
Tissue blocks/filters completely surrounded by gelatin will cut easier

48

Flat embedding 2
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.

Grow cells on coverslips or in dishes (with or without coating)

Put the coverslip in the 3 cm dish, cells up
Fix the cells (cells fixed in 2% PFA/0.2% GA detach better then 4% PFA alone)
Rinse 3 x with PBS
Rinse 10 min. with PBS/Glycin
Rinse briefly with PBS/0.1% BSA, this facilitates spreading of gelatin
Remove the PBS/BSA and directly put on a thin layer (800 l) of 12% gelatin
37, gently swirl the dish to spread the gelatin
Incubate the dish in a 37C stove for 30 min. (dish in a wet chamber!)
Solidify on room temperature 3
Rinse briefly with PBS/0.1% BSA
Remove the PBS/BSA and directly put on a thin layer (400 l) of prewarmed
37C 12% gelatin + fixed erythrocytes on the dish, gently swirl the dish to
spread the gelatin
Solidify at 4C
Fix the gelatin with 0.5-1% PFA 30 min. at 0 C (ice)
Rinse 3x with cold PBS
Add cold 2.3 M sucrose on top
Place the dish at 4C (cold room) on a rocker
Replace the sucrose for fresh cold sucrose after 30 min.
Place the dish at 4C (cold room) on a rocker at for a few days

Yeast
1.
2.
3.
4.
5.

6.
7.
8.
9.

2
3

Wash the cells 3x in 0.1M PHEM buffer.

Between each wash, briefly centrifuge in a minifuge (few seconds).
Add 1% of freshly-prepared Periodic Acid in 0.1M PHEM buffer (the periodic
acid will cleave the sugar groups causing the cell wall to stay in place.).
Incubate at room temp for 1 hour on a roller bank or a rotating wheel at a
slower speed.
Again, wash 3x with 0.1M PHEM buffer with a brief centrifuge time in the
minifuge.
(Yeast cells tend to clump together after this stageyou can try to
resuspend them by means of a Pasteur pipette but the chances are that
some clumps will remain.).
After the last rinse, remove all the buffer (dont let the cells fall dry!) and add
12% gelatin in 0.1M PHEM buffer.
Incubate the cells at 37C for 5-10 minutes (this step is necessary to make
sure that the yeast clumps are properly infiltrated).
Cool the samples down on ice (if you run out of time, you can keep the sample
in gelatin at 4C overnight).
Cut blocks of 1 mm3.

Due to the different sectioning properties of cells and filter the cells are often broken away
from the filter during sectioning. This problem can partially be overcome by using a
See also van Rijnsoever, Oorschot et al. 2008

4C is to cold, the 2nd layer of gelatin wont stick.

49

11.6.

Immuno-labeling

Materials and equipment

1.
2.
3.
4.
5.
6.
7.

37 C stove
Filter paper Whatman 50
Hood
Parafilm
Remanium wire loops 3.5-4 mm
Transport plate for grids
Tweezer

Reagents
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.

2% gelatin plates
PBS
PBS/0.15% Glycine (20 mM)
PBS/0.1% BSA
PBS/1% BSA
10% BSA
1% Glutaraldehyde (GA) in PBS
2% Uranyloxaalacetate PH7 (UOA)
Methyl-cellulose/Uranyl acetate PH4 (MC/UA)
Protein-A-Gold (PAG)

For immuno-fluorescence labeling also:

1.
Mountin medium [Vecta shield: Vector laboratories H-1200; Prolong gold
:Invitrogen P-7481]
2.
Microscope slide
3.
coverslips
Procedure
Single labeling
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
1

Place the grids, sections down on a 2% gelatin plate

Put the plate in a wet chamber (e.g. a 9 cm Petri dish with wet filter paper)
Melt the plate under a lamp
Keep the plate in a 37 C stove for
20-30 min.
(Removal of the MC/Sucrose and the 12% gelatin in-between the cells) 1
PBS/Gly
3 x 2 min.
(75 l drops on parafilm at room temperature)
PBS/1% BSA
5 min.
Antibody diluted in PBS/1% BSA
30-120 min.
PBS/0.1% BSA
5 x 2 min.
Bridging antibody in PBS/1% BSA
20-30 min.
PBS/0.1% BSA
5 x 2 min.
Protein-A-Gold (PAG) in PBS/1% BSA
20-30 min.
PBS 5 x 2 min.

Insufficient rinsing of gelatin leads to a gelatin layer over the sections and will influence the
labeling quality

50

15.
16.
17.
18.
19.
20.

1% GA in PBS (in fume-hood) 1,2

Distilled water
2% Uranyloxalate-acetate PH7 3
MC/UA pH4 on ice
MC/UA pH4 on ice 4
Dry the grid by the looping out method

5 min.
6 x 1 min.
5 min.
2x briefly
5-10 min.

Looping out method

1.
2.
3.
4.
5.
6.

Clean a remanium wire loop with distilled water

Push the loop into the MC/UA droplet at some distance from the floating grid
Move the loop under the grid
Lift the grid from the MC/UA droplet
Tilt the loop and grid to an angle of 45-60, the excess MC/UA downwards
Touch the side of the loop and grid to well absorbing filter paper with the
sections facing the filter paper
7.
As soon as the MC/UA touch the paper move the grid gently sideways until the
removal of MC/UA stops
8.
A thin even film of MC/UA remains on the surface of the grid 5
9.
Dry the grid for at least 30 min.
10. The section is visible as a bluish islet in a golden sea
11. Remove the grid form the loop with a fine tweezer and store in a gridbox
Double labeling
1.
2.
3.
4.
5.

2
3
4
5

6,7,8,9

Perform step 1-15 from single labeling

PBS 2 x 1 min.
PBS/Gly
Follow step 8-20 from single labeling
Use a different size of PAG

5 x 2 min.

This rinsing step is very important with the flat embedded fixed gelatin specimen sections.
Rinsing away need to be done at 37C in PBS for at least 1h.
GA in water can dissociate PAG from the antibody, therefore we use GA in buffer.
UOA pH 7 can be omitted (e.g. dense tissue)
If the contrast is too low, increase the concentration of UA in the MC
MC too thin: sections easy visible in EM, blurry, no sharp membranes, holes in cytoplasma
MC too thick: sections embedded in a dense gray MC layer, hardly visible in EM, image not
blurred
Possible to label 2 polyclonals in combination with protein A-Gold or 1 polyconal and 1
monoclonal (different species)
Not possible to label 2 monoclonals because the bridging steps will cause false positives
(even after GA step)
Before performing a double labelling first do a single labelling with both antibodies to get
acquainted with the labelling pattern.
Try each primary antibody as the first ab in combination with the different markers

51

Immuno-fluorescence labeling
1.
2.
3.
4.
5.
6.
7.

Encircle the sections with wax-pen

Put the slides in a wet chamber
Put melted 2% gelatin on the sections and incubate at 37C for 30 min.
Follow the labeling procedure as mentioned in single labelingReplace the PAG
by a fluorochrome coupled secondary IgG
Rinse with distilled water 5 x 2 min.
Nearly dry the surface
Cover the slide with mounting medium and a coverslip

52

11.7.
Ionizer (Static Line II) usage
The ionizer is an anti-static device, it emits positive and negative ions and
neutralizes the electrostatic charge in the cryochamber. The ionizer is mainly used
with diamond knives, but can be helpful with glass knives as well
Usage during sectioning:
Try to prevent sticking of the very first sections to the cutting-edge of the diamond
knife by:
1.
2.
3.
4.
5.
6.

Start sectioning with the ionizer at position 9.

Reduce the voltage after a few sections.
If sections lift up from the knife surface: reduce the voltage.
If sections stick at the cutting edge: increase the voltage.
Switch ionizer to position 1 or 2 or even off during transfer of the sections with
a hair.
Switch the ionizer always off during pick-up of the sections.

Helpful hints:
1.

The ionizer will lose its power if the tip becomes covered with ice. If this
occurs, just clean the tip with a fine brush.
2.
Heavy ice-crystal growth on the ionizer causes very irregular cutting. Turn it
off and take the ionizer out of the cryochamber, let it thaw and dry the
ionizer with a hair dryer.
3.
The ionizer switched to the highest position ( position 10) can influence the
cutting window and on/off switch. Do not use the ionizer at a setting above
9.
4.
Check always if the tip of the ionizer is in line with the knife-edge.
5.
To avoid breaking, do not bend the frozen cable.
6.
Switch off the unit prior to doing any manipulation in the chamber (specimen
change, knife change, etc).
7.
When the ionizer is on, the tip of the ionizer should not come in contact with
the metallic parts of the chamber.
8.
Never heat the chamber with the ionizer inside.
9.
Electrostatic charging in the chamber is dependent on different factors such as
chamber temperature, humidity, specimen, PFA-fixation or GA-fixation of
the specimen, cutting speed , shoes and clothes, LN2 flow in the
cryochamber, etc. It may be necessary to vary the ion emission as the
sectioning conditions change.
10. Switch off the unit before taking the ionizer from the cryochamber.
How to check the electrode of the ionizer with the multicheck:
According to Helmut Gngi:
Test the electrode with the Multicheck tester as follows: Place the electrode in the
cryochamber and test at low temperature(-110 or -120C). Approach the electrode
tip with the black nose of the Multicheck, while pressing the button. At first the diode
at the Multicheck is red, when coming closer it turns to green. For perfect function
the green should be lit at approximately 20 mm. Old electrodes often turn green at
approximately 5 mm. An old electrode has only a very small effect on section gliding.
The metal tips of the electrodes erode per time used. It is advisable to buy a new
one after may be two years.
53

11.8.

Mounting & Freezing

Materials and equipment

1.
2.
3.
4.
5.
6.
7.
8.
9.

Binoculars
Double edge razor blade (necessary with flat embedding)
Filter paper
LN2 container
Scalpel (necessary with flat embedding)
Sharp metal point or sandpaper
Sonicator
Specimen carriers copper or aluminium (pins)
Tweezers

Reagents
1.
2.
3.

Aceton or 100% ethanol

2.3M sucrose
2.3M sucrose in 1% toluidine blue first make 1% toluidine blue solution than
dissolve the sucrose

Procedure
1.
2.
3.
4.

Preparation of the specimen carriers

Roughen the top of the pins with a sharp metal point or sandpaper 1
Sonicate the pins in aceton or 100% ethanol
Check with binoculars whether the pins are perfectly clean and free of metal
remnants

Mounting & freezing

The mounting/freezing procedure should be done as quickly as possible. Water
evaporates from the specimen during air exposure, and then sucrose concentration
becomes so high that sucrose crystals will be formed. We usually do this part under
a binocular in the cold room. If you do not have a cold room available, be quick, use
ice and freeze your blocks one by one.
1.
2.
3.
4.
5.

Remove a specimen block from the 2.3M sucrose

Place the block on the table of the clean pin
Remove most of the excess sucrose with a piece of filter paper
Mount the block in the right orientation 3
Freeze the pins/blocks in a LN2 r 4

Recommended literature: Slot and Geuze 2007.

Metal remnants will damage your knife extremely, take care, clean the pins very
well and check
2
If there is too much sucrose left, the blocks will easily break of the pins after
freezing
3
Orientate elongated cells in a vertical position (cutting surface), it will cut easier
4
If the blocks are big, slowly freeze them in the cryo chamber before putting them in
LN2
54

Mounting & freezing Flat embedded specimen

1.
2.
3.
4.
5.
6.
7.
8.

After sucrose infusion scarify the gelatin with a scalpel

Take off a slab of gelatin/cells
Stain the slabs in 2.3M sucrose in 1% toluidine blue for 1h at 4C
Rinse in white 2.3M sucrose, the cells will stay blue the gelatin becomes
clear 1
Make small blocks where cells are located with a double edge razor blade
Mount the blocks on the pins as above 2
Slowly freeze the pins in the cryo chamber
Store the pins/blocks in a LN2 container

Storage
Frozen specimens (on specimen holders) can be stored in LN2 for months or even
years. 3

Toluidin penetrates gelatin faster than cells so first over stain the gelatin/cell block, then
rinse away till the gelatin becomes clear while the cells still contain toluidin blue
2
Flat embedded cells can be mounted flat or perpendicular to the pins (easy to see the
polarity)
3
If, after a long storage time , the blocks become to brittle, re-infuse them with sucrose

55

11.9.

Loops and hairs preparation

Materials:
1.
Bamboo sticks
2.
Dalmatian hairs (or your own)
3.
Epon resin
4.
Mini vice
5.
Nail polish
6.
Pipette tips
7.
Razor blades
8.
Remanium wire (soft stainless steel orthodontists wire )

Figure 11.5: Loops and hair dimensions

56

Pickup loop:
1.
2.
3.
4.

Make a 2.5 mm wire loop of 0.3 mm soft stainless steel, the length of the
shaft should be about 1.5 cm long. Cut the rear end.
Cut off a small part of a yellow pipette tip and fill with approximately 50
microliter resin (e.g. EPON).
Press the shaft of the wire loop in the opening of the tip for 0.5 cm and push a
wooden stick into the other end.
Let the EPON polymerize at 80 C overnight. (Optional: remove the excess
part of the pipette tip).

Drying loop
1.
2.
3.
4.

make a 4 mm wire loop of 0.3 mm soft stainless steel, the length of the shaft
should be about 1.5 cm long. Do not twist too much and cut the rear end.
fill a blue pipette tip with approximately 200 microliter resin
Press the shaft of the wire loop in the opening of the tip for 1 cm
Let the EPON polymerize at 80 C overnight. (Optional: remove the excess
part of the pipette tip).

Hair:
1.
2.
3.
4.
5.
6.

Split a bamboostick with a razor blade on the top.

Leave the tip of the razor blade in the top of the
stick in such a way that the very top is free.
Put a human eyebrow hair or Dalmatian hair (or
species of choice) on adhesive tape and cut to 812 mm
Put a bit of nail polish on the split top and insert a
hair
Remove razor blade.
Check position of the hair under binocular, correct if
necessary
Figure 11.6: Genuine
Dry.
Dalmatian

57

7.
11.10.

Protein A-Gold (Tannic acid procedure)

Materials and equipment

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.

big magnet
centrifuge
centrifuge swing-out rotor
electron microscope
erlenmeijers
glass beakers
glass vial 5 ml with stirring bar
gloves
gradint pomp, mixer, tubes
graduated cylinders
grids and tweezers
magnetic stirrers
magnetic stirring bars
pH meter
plastic lids for glass beaker (e.g. plastic culture dish)
Small transparent test tubes
spectrophotometer
thermometers
vacuum pomp with short glass pipet

Reagents
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.

[Sigma G4022-IG]
1% HAuCl4 -3H2O Gold (III) Chloride trihydrate
1% Citrate tri-Na-Citrate-dihydrate [Merck 1.06448.1000[
Freshly prepared1% Tannic acid; Mallincrodt AR (Aleppo)
0.2 mg/ml Proteine A; [GE-healthcare Bioscience AB 17-0872-50]
0.1M NaOH freshly made
10% NaCl
10% BSA [Sigma A9647 Fractio V]
10% Na-azide
10% glycerol prepare 1 day before [J.T. Baker 7044]
30% glycerol prepare 1 day before
H2O

All the solutions are made in distilled water (again filtered and deionised through a
Millipore filter unit)
Procedure
Cleaning the glassware
Reagents setup
Preparation of a gold sol
pH adjustment
Proteine A binding to gold
Purification of the PAG complex
OD measurements

58

Cleaning the glassware before and afterwards:

1.
2.
3.
4.

Wash the glassware, stirring bars en thermometers with water and soap
Rinse with tap water
Boil with distilled water or put boiling distilled water into the graduated
cylinders and small glassware
Cool till room temperature before use

Reagents setup:
1% HAuCl4 -3H2O
1.
2.
3.
4.
5.

Use a glass beaker without stirring bar

Dissolve 1 gram in 100 ml AD by shaking
Store at 4C, light sensitive!
Filter through a 0.33 m milipore filter before use
Warm till room temperature before use

1% Citrate buffer
6.
7.
8.
9.

Use an Erlenmeyer without stirring bar

Dissolve 1 gram in 100 ml AD by shaking
Store at 4C
Warm till room temperature before use

1% Tannic acid
Make
1.
2.
3.
4.

fresh
Use a 25 ml beaker with stirring bar
Put 20 ml AD in the beaker
Add 0.2 gram TA
Cover with Parafilm and stir till dissolved

Note: The Aleppo tannic acid we use is no longer available. Tannic acid is purified
from many different plant sources, each with its own impurities. Use tannic acid
purified from one kind of plant (when available) for reproducible results.
5 mg/ml Proteine A
1.
2.
3.
4.
5.

Use a beaker without stirring bar

Add 1 vial of Prot. A (50 mg)
Add 250 ml AD and dissolve by shaking
Store at - 20C
Thaw before use

0.1M NaOH
Make a 10 x dilution from 1M NAOH
1M NaOH
Dissolve 0.4 gr NaOH in 10 ml distilled water (final volume), stir
59

10% NaCl
Dissolve 1 gram in 10 ml distilled water (final volume), stir
10% BSA
1.
2.
3.
4.
5.

Put 10 gram BSA in a beaker with stirring bar

Add distilled water till 100 ml
Stir gently (prevent air bubbles)
Centrifuge for 75 min at 30.000 RPM
Aliquot and store at - 20C

10% Na-azide
1.
2.

Add 1 gram in 10 ml distilled water, stir

Aliquot and store at - 20C

10% Glycerol in PBS

3.
4.
5.
6.
7.
8.
9.
10.

Use a graduated cylinder of 250 ml

Add 222 ml PBS
Add 25 ml 100% glycerol
Shake very well
Add 2.5 ml 10% BSA
Shake gently
Add 0.5 ml 10% azide
Store at - 4C

30% Glycerol in PBS (3 x 250 ml)

1.
2.
3.
4.
5.
6.
7.
8.

Use a graduated cylinder of 250 ml

Add 172 ml PBS
Add 75 ml 100% glycerol
Shake very well
Add 2.5 ml 10% BSA
Shake gently
Add 0.5 ml 10% azide
Store at - 4C

60

Preparation of gold sol:

As an example the procedure for the preparation of 15 nm gold is given. The other
sizes require different reagent and spinning times and G forces as stated in table 111.
Table 11-1: Requirements for different gold sizes and the used dilutions.
nm size

l 1% TA

l 25mM
K2CO3

Centrifuge

Gradient
centrifuge

3
5
10
15
20

2500
580
75
15
grown from 15

2500
0
0
0
0

41.400 G 1h
41.400 G 1h
25.300 G 1h
18.700 G 1h
8.700 G 1h

89.500
89.500
42.900
19.100
10.700

G
G
G
G
G

75
75
45
30
20

Use OD
min.
min.
min.
min.
min.

0.1
0.1
0.2
0.3
0.3

Preparation of 15nm gold sol (example)

Solution A: Gold-chloride solution (HAuCl4)
1.
100 ml beaker with thermometer and stirring bar
2.
Add 79 ml Milli Q water
3.
Add 1 ml 1% HAuCl4 (filtered)
SolutionB: Reducing mixture (TA)
1.
50 ml beaker with thermometer and stirring bar
2.
Add 16 ml Milli Q water
3.
Add 4 ml 1% NaCitrate
4.
Add 15 l 1% TA
Procedure
Preparation of the gold sol
1.
2.
3.
4.
5.
6.
7.
8.

Heat solution A and B to 60C

Add B quickly to A while stirring vigorously
Red sols are formed within a second when high concentrations of TA are
added. The reaction time increases gradually when lower concentrations of
TA are used
Slowly stir the sol at 60C till the color changes from purple to red
Heat the sol till boiling
Cool down on ice to room temp. while covered with a plastic lid
Check the sol quality in the EM
Cover with parafilm, store at 4C

pH adjustment:
1.
2.
3.
4.
5.

Bring sol to room temperature

Take 5 ml sol
Add 125 l Proteine A 0.2 mg/ml
Stir 5 min.
Measure the pH and adjust to pH 6.1 with 0.1N NaOH

61

6.
7.

Calculate the volume of NaOH for the total sol volume

Just before protein-A binding : adjust sol pH to pH 6.1 with 1M NaOH

Proteine A binding to gold:

1.
2.
3.
4.

Add 250 l sol pH 6.1 into test tubes

Add 0.2 mg/ml Prot. A in a range of 0.25 2.50 g protein per tube, stir
After 1 min. add 25 l 10% NaCl
The lowest protein concentration that prevents the red to blue colour change
from occurring (which can be judged visually) is taken as the stabilization
concentration, and from that value the gold number can be calculated.
5.
Calculate the volume of Protein A for the total sol volume
6.
Add the protein A to the sol while stirring slowly
7.
Stir 20 min.
8.
Adjust the pH to 7.3 7.5 with NaOH
9.
Add bovine serum albumin (BSA), at a final concentration of 0.1%, to be sure
that the gold particles are stabilized maximally
10. Take the stirring bar out and store in the cold room

Purification of the PAG complex:

1.

2.
3.
4.
5.
6.
7.
8.

Spin the gold particles down: PAG 15: 1h 18,700 xg at 4C

The resulting pellet is composed of a large loose part and a small tightly
packed part. If the centrifugation force is too high the tight pellet becomes
too large and an increasing amount of aggregates of gold particles is
introduced into the preparation.
Remove the supernatant without disturbing the pellet and
Resuspend the loose part of the pellet in a small volume
Purify the concentrated PAG probe by layering it over a 10 - 30% continuous
glycerol gradient in 0.1% BSA/PBS (see reagents setup)
Centrifuge the gradient ; PAG15: 30 min, 19,500 xg at 4C(use a swing out
rotor)
Collect the dark red band. It contains essentially a mono-disperse PAG
preparation without free protein A.
Add Na-azide to an end concentration of 0.02%
Store at 4C

OD measurements:
1.
2.
3.

Dilute the PAG 1:50 with PBS

Measure the Optical Density (OD) in a Spectrophotometer at =520 nm
against PBS
Calculate the dilution for immuno-EM labeling (see table 11-1)

62

11.11.

Silan coated microscope slides

Materials and equipment

1.
2.
3.
4.
5.

Microscope slides
Glass microscope slide rack
Glass vial
Filter paper
57C stove

Reagents
1.
2.

Silan: 3-aminopropyltriethoxysilan
Ethanol 100%

Procedure
1.
2.
3.
4.
5.
6.

Make a mixture of silan and ethanol 1:19 in a glass vial

Put the clean microscope slides in the mixture for 1 min.
Transfer the slides into distilled water, rinse 2 x 1 min.
Place them vertically on filter paper and let the excess of water absorb
Dry the slides for 3 hrs at 57C
Store at room temperature, no limit

63

11.12.

Section Light Electron Microscopy (SLEM) Immuno-labeling

Materials and equipment

1.
2.
3.
4.
5.
6.
7.

37 C stove
Parafilm
Tweezer
Remanium wire loops 3.5-4 mm
Filter paper Whatman 50
Fume-hood
Finder grids

Reagents
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.

2% gelatine plates
PBS
PBS/0.15% Glycine (~20 mM)
PBS/0.1% BSA
PBS/1% BSA
10% BSA
1% Glutaraldehyde (GA) in PBS
2% Uranyloxaalacetate PH7 (UOA)
Methyl-cellulose/Uranyl acetate PH4 (MC/UA)
Protein-A-Gold (PAG)
50% Glycerol in Distilled water

Procedure
SLEM labeling
1.
2.
3.
4.
5.

Place the finder grids, sections down on a 2% gelatine plate

Put the plate in a wet chamber (e.g. a 9 cm Petri dish with wet filter paper)
Melt the plate under a lamp1
Keep the plate in a 37 C stove for 20-30 min.
Removal of the dried MC/Sucrose and the 12% gelatine in-between the cells

Unless indicated differently, the labeling will take place at room temperature on
parafilm with droplets of the solution mentioned and grids floating on top:
6.
7.
8.
9.
10.
11.

PBS/Gly
PBS/1% BSA
Antibody diluted in PBS/1% BSA
PBS/0.1% BSA
Bridging antibody in PBS/1% BSA
PBS/0.1% BSA

3x2
5
30-120
5x2
20-30
5x2

min.
min.
min.
min.
min.
min.

Insufficient rinsing of gelatine leads to a gelatin layer over the sections and will influence
the labelling quality
This rinse step is very important with the flat embedded fixed gelatine specimen sections.
Rinsing away need to be done at 37C in PBS for at least 1h

64

12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.

Rabbit IgG / Alexa 488 in PBS/1% BSA

PBS/0.1% BSA
Protein-A-Gold (PAG) in PBS/1% BSA
PBS
1% GA in PBS 1
PBS
Hoechst 33342 (1 g/ml) in PBS 2
PBS
Distilled water
Mount grid between object slide and coverslip (24x60):
Dip the grid in 50% glycerol in water
Put grid on thin object slide, sections facing up
Add droplet of glycerol
Cover with coverslip
IF on the Deltavision Light microscope
Select the cell of interest
Make an overview picture at 40x
Make a detail picture at 100x
Do the same with other cells of interest
Remove the oil carefully from the coverslip
Unmount the grid in distilled water
Dry the backside of the grid
Rinse in distilled water
Put the grid on droplets, sections down:
2% Uranyloxaalacetate PH7
MC/UA pH4
MC/UA pH4
Dry the grid by the looping out method 3

45 min.
5 x 2 min.
20-30 min.
5 x 2 min.
5 min.fume-hood
5 x 2 min.
20 min.
2 x 2 min.
3 x 2 min.

8 x 2 min
5 min.
2x briefly on ice
5-10 min.on ice

Looping out method:

1.
2.
3.
4.
5.
6.

Clean a remanium wire loop with distilled water

Push the loop into the MC/UA droplet at some distance from the floating grid
Move the loop under the grid
Lift the grid from the MC/UA droplet
Tilt the loop and grid to an angle of 45-60, the excess MC/UA downwards
Touch the side of the loop and grid to well absorbing filter paper with the
sections facing the filter paper
7.
As soon as the MC/UA touch the paper move the grid gently sideways until the
removal of MC/UA stops
8.
A thin even film of MC/UA remains on the surface of the grid 4
9.
Dry the grid for at least 30 min.
10. The section is visible as a bluish islet in a golden sea
11. Remove the grid form the loop with a fine tweezers and store in a gridbox
See also Cortese et al(2009)
1
2
3
4

GA in water can dissociate PAG from the antibody, therefore we use GA in buffer
UOA PH 7 can be left out (e.g. dense tissue)
If the contrast is too low, increase the concentration of UA in the methyl cellulose
MC too thin: sections easy visible in EM, blurry, no sharp membranes, holes in cytoplasma
MC too thick: sections embedded in a dense gray MC layer, hardly visible in EM, image
not sharp

65

12.

Recipes

12.1.

BSA 10% (100 ml)

Materials and equipment

1.
2.
3.
4.
5.
6.

Centrifuge
erlenmeyer of 100 ml
graduated cylinders
Magnetic stirrer
pH meter
stirring bar

Reagents
1.
2.
3.

Bovine Serum Albumin Fraction V, [Sigma A-9647]

Na-Azide 20% (2g in 10 ml H2O) [ Merck 822335]
H2O

Procedure
1.
2.
3.
4.
5.
6.
7.

Add 10 g in 60 ml distilled water (Milli Q) while stirring slowly

stir slowly (to prevent foaming) overnight at 4C
Adjust the pH 7.4 to with 1N NaOH
Add 100 l 20% azide
Add distilled water till 100 ml, stir gently
Centrifuge for 1h at 100.000 G
The supernatant is stored in small aliquots at 4C

12.2.

Formaldehyde 16% (500 ml)

Materials and equipment

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.

Beaker 2 liter
Bunsen burner
Erlenmeyer 500 ml
Filter paper
Fume hood
Funnel
Gloves
Graduated cylinder 500 ml
Magnetic stirrer/heater
pH indicator stick
Stirring bar
Thermometer

Reagents
1.
2.

NaOH (1M in dist. water)

PFA (Paraformaldehyde) prills 95% [Sigma/Aldrich441244]

66

Procedure
1.
2.
3.
4.
5.
6.
7.
8.
9.

Add 80 g PFA in 400 ml distilled water in an Erlenmeyer + thermometer

Place the solution au bain marie (in another beaker with hot water) on a
combined stirrer/heater in the hood
Stir for 15 min 60 C NOT warmer
Add 0.1M NaOH till pH 7, check with pH paper.
Stir for 30 min 60 C NOT warmer (at this point the solution is not clear)
Cool till room temperature, check pH 7
Add distilled water till 500 ml
Filter the solution (it becomes clear)
Freeze in aliquots

Each time that PFA fixative is required a tube can be thawed and used. Upon thawing
the solution will remain white. Keep the PFA-vial then in hot (tap) water till it gets
clear again. Do not use the solution if it does not get clear.
12.3.

Formvar solution for grids (100 ml)

Materials and equipment

1.
2.
3.
4.

Fume hood
Magnetic stirrer
stirring bar
volumetric flask with stopper 100 ml

Reagents
1.
2.

Choroform analytical grade

Formvar 15/95 E [Vinylec E, SPI-chem 02492-RA]

Procedure
1.
2.
3.
4.
5.
6.
7.
8.
9.

Clean the volumetric flask with stopper and the stirring bar with acetone
followed by chloroform
Dry the flask and stopper
Weigh 1.1 g Formvar in the flask
Add chloroform till full (in the hood)
Stir until dissolved (10-20 min.)
Remove the stirring bar
Add chloroform till 100 ml
Let the solution rest overnight in dark place at room temperature
Store in dark place at room temperature (shelf life approx. 2 months,
depending on how many times the flask has been openend)

Caution: Chloroform is toxic, work in a hood.

67

12.4.

gelatin 2% in 3cm Petri dish (100 ml)

Materials and equipment

1.
2.
3.
4.

Beaker of 100 ml
Magnetic stirrer
Petri dishes3 cm
Stirring bar

Reagents
1.
2.
3.
4.

Gelatin powder food quality

Na-Azide 20% (2g/10 ml dist. water)
Phosphate buffer 0.1M
H2O

Procedure
1.
2.
3.
4.
5.
6.
7.
8.
9.

Add 2 g gelatin in a 75 ml 0.1M Phosphate buffer

Stir 10 min. at room temperature
Warm the solution at 60C for 4-6 hrs (no stirring needed)
When the gelatin has dissolved cool the solution till 37C
Add 100 l 20% azide
Add 0.1M Phosphate buffer till 100 ml, stir gently
While still at 37C pour 3 ml into 3 cm petri dishes (use lid and bottom)
Put the filled 3 cm dishes in a 10 cm Petri dish
Cool in the refrigerator with the lid of the 10 cm slightly open, close the dish
the everything has cooled down completely
10. Store in tight fitting containers at 4C

12.5.

Gelatin 12% for cell support (100 ml)

Materials and equipment

1.
2.
3.
4.

Erlenmeyer of 100 ml
Magnetic stirrer
Stirring bar
Vials 75 ml

Reagents
1.
2.
3.

Gelatin powder, food quality

Na-Azide 20% (2g/10 ml dist. water)
Phosphate buffer 0.1M,

Procedure
1.
2.
3.
4.
5.
6.
7.

Add 12 g gelatin to 75 ml 0.1M Phosphate buffer

Stir 10 min. at room temperature
Warm the solution at 60C for 4-6 hrs (stir gently)
When the gelatin has dissolved cool the solution till 37C
Add 100 l 20% azide
Add 0.1M Phosphate buffer till 100 ml, stir gently
Store in 7 ml vials at 4C

68

12.6.

Methyl cellulose 2% (200 ml)

Materials and equipment

1.
2.
3.
4.
5.
6.

Centrifuge
Erlenmeyer 250 ml
Graduated cylinder 200 ml
Magnetic stirrer
Stirring bar
Tubes (10 ml) with a screw cap

Reagents
1.
2.

Methyl cellulose 25 centipoises: [Sigma M-6385]

H2O

Procedure
1.
2.
3.
4.
5.
6.
7.
8.
9.

Heat 196 ml distilled water to a temperature of 90C

Add 4 g methyl cellulose while stirring
Rapidly cool the solution on ice while stirring
Seal with parafilm and stir overnight at 4C
Stop stirring, let the solution ripen for 2 days at 4C
Add distilled water till 200 ml
Centrifuge for 1.5 hrs at 100.000 G
Pour the supernatant in tubes with a screw cap
Store at 4C in the dark (shelf life: 6-9 months)

12.7.

Methyl celluloseuranyl acetate pH 4 (10 ml)

Materials and equipment

1.
2.
3.

Tube 10 ml with screw cap

Syringe 1 ml
Milipore 0.45 m filter

Reagents
1.
2.

2% Methyl cellulose in AD
4% Uranyl acetate in AD

Procedure
1.
2.
3.

Add to 9 ml of the methyl cellulose 1 ml of the uranyl acetate solution

(Milipore filtered)
Mix gently
Store at 4C in the dark (shelf life: 3 months)

69

12.8.

PBS 10X pH 7.3 (2.5 lit.)

Materials and equipment

1.
2.
3.
4.

Erlenmeyer of 2.5 liter

Graduated cylinder
pH meter
Stirring bar

Reagents
1.
2.
3.
4.
5.

NaH2PO4. 1H2O,
Na2HPO4. 2H2O
NaCL
KCL
H2O

Procedure
1.
2.
3.
4.
5.
6.
7.
8.

Add to 1500 ml distilled water:

5.75 g NaH2PO4. 1H2O
36 g Na2HPO4. 2H2O
200 g NaCL
5 g KCL
Stir , remove stirrer bar and add distilled water till 2500 ml
Check the pH 7.3
Store at room temperature

12.9.

PBS/glycine 0.02 M

Materials and equipment

1.
2.

Erlenmeyer of 100 ml
Balance

Reagents
1.
2.
3.

H2O
PBS
Glycine

Procedure
1.
2.
3.
4.

Pour 10 ml 10x PBS in a 100 ml Erlenmeyer

Add 0.15 g glycine
Swirl till glycine is dissolved
Add H20 till 100 ml

70

12.10.

PHEM, 0.2M buffer pH 6.9 (100ml)

Materials and equipment

1.
2.
3.
4.
5.

Erlenmeyer of 100 ml
Graduated cylinder
Magnetic stirrer
pH meter
Stirring bar

Reagents
1.
2.
3.
4.
5.

EGTA [Merck 324626]

Hepes [Merck 391340]
MgCl2. 6H2O
NaOH 1M (0.4g/10 ml dist. water)
Pipes (Piperazin-1,4-bis (2 ethansulfonsure) [Merck 110220]

Procedure
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.

Add 3 pellets of NaOH in 75 ml distilled water, stir

Add 3.63 g Pipes (120 mM), stir at room temperature till the solution is clear
Check the pH (~pH 7)
Add 1.19 g Hepes (50 mM), stir
Add 0.08 g MgCL2 (4 mM), stir
Add 0.76 g EGTA (20 mM), stir
Adjust the pH 6.9 with 1M NaOH
Add distilled water till 100 ml
Store at 4C or freeze in aliquots

12.11.

Phosphate buffer 0.2M pH 7.4 (100 ml)

Materials and equipment

1.
2.
3.
4.
5.

Erlenmeyers (3) of 100 ml

Graded cylinders (2)
Magnetic stirrer
pH meter
Stirring bars (3)

Reagents
1.
2.
3.

NaH2PO4. 1H2O
Na2HPO4. 2H2O
H2O

Procedure
Stock A: 0.2M NaH2PO4.1H2O
1.
2.
3.

Add 2.76 g in 70 ml distilled water, stir

Add distilled water till 100 ml
Store at room temperature

71

Stock B : 0.2M Na2HPO4. 2H2O

1.
2.
3.

Add 3.56 g in 70 ml distilled water, stir

Add distilled water till 100 ml
Store at room temperature

0.2M Phosphate buffer pH 7.4

Before use
1.
Add to 19 ml stock A 81 ml stock B, stir
2.
Check the PH
12.12.

Pipes 0.2M buffer pH 7.3 (200ml)

Materials and equipment

1.
2.
3.
4.
5.

Erlenmeyer of 200 ml
Graduated cylinder 200 ml
Magnetic stirrer
pH meter
Stirring bar

Reagents
1.
2.

Pipes (Piperazin-1,4-bis (2 ethansulfonsure) [Merck 110220]

NaOH

Procedure
1.
2.
3.
4.
5.

Add 12.09 g Pipes in 150 ml distilled water

Add 2 g NaOH, stir at room temperature till the solution is clear
Adjust the pH 7.3 with 1M NaOH
Add distilled water till 200 ml
Store at 4C or freeze in aliquots

12.13.

PVP - Sucrose pH 10 (20 ml)

Materials and equipment

1.
2.
3.
4.

Aluminium foil
Beaker 50 ml
Magnetic stirrer
Stirring bar

Reagents
1.
2.
3.
4.

H2O
Na2CO3
PVP; Polyvinylpyrrolidone Mw 10.000 [Sigma PVP-10]
Sucrose ; D(+) Sacharose

Procedure

72

1.
2.
3.
4.
5.
6.

Make a marker point of 20 ml in the beaker + stirring bar

Mix in the beaker 3 g PVP, 11.62 g sucrose, 0.69 g Na2CO3
Add distilled water till the marker point
Wrap in aluminum foil (light sensitive)
Stir for 3 hrs
Store at 4C in the dark

The solution can also be made in the buffer of choice (pH will be different)
12.14.

Sucrose 2.3M (100 ml)

Materials and equipment

1.
2.
3.
4.

Beaker 100 ml
Magnetic stirrer
Stirring bar
Vials 1 ml

Reagents
1.
2.

Sucrose D(+) Saccharose

0.1M phosphate buffer(see recipes)

Procedure
1.
2.
3.
4.
5.
6.

Make a marker point of 100 ml in the beaker + stirring bar

Add 78.73 g sucrose
Add 0.1M phosphate buffer till the marker point
Stir until the sucrose is dissolved
Aliquot in 1 ml vials
Store at 4C

12.15.

Uranyloxalicacetate PH7 (100 ml)

Materials and equipment

1.
2.
3.
4.
5.
6.

Erlenmeyers 100 ml (3)

Magnetic stirrer
Milipore 0.45 m filter
pH indicator sticks
Stirring bars
Syringe

Reagents
1.
2.
3.
4.

Ammonium hydroxide 25% NH4OH

H2O
Oxalic acid (2 H2O)
Uranyl acetate [SPI-chem 02624-AB]

73

Reagents set up
4% uranyl acetate
1.
2.
3.
4.

Add 4 g uranylacetate in 90 ml distilled water

Stir at room temperature in the dark till dissolved (2 hrs)
Remove the stirring bar
Add distilled water till 100 ml

Oxalic acid)
1.
2.
3.
4.

Add 3.8 g oxalic acid in 90 ml distilled water

Stir at room temperature till dissolved (2 hrs)
Remove the stirring bar
Add distilled water till 100 ml

Procedure
2% UA in 0.15M
1.
2.
3.
4.
5.
6.
7.

Add 50 ml 4% uranylacetate to 50 ml oxalic acid, stir

Add NH4OH drop by drop to PH7 while constantly stirring
Check with a pH indicator stick
If too much NH4OH is added the pH exceeds 8 and precipitates. Do not use
this UOA!!!
If the NH4OH is too old (too much CO3- in the bottle) the uranyloxalicacetate
will be neutral and will give less contrast and possible precipitates
Filter through a 0.45 m Millipore filter
Store at 4C in the dark

12.16.

Uranylacetate 4% pH4 (10 ml)

Materials and equipment

1.
2.
3.
4.
5.

Erlenmeyer 10 ml
Stirring bar
Magnetic stirrer
Milipore 0.45 m filter
syringe

Reagents
1.
2.

Uranyl acetate [SPI 02624-AB]

H20

Procedure
1.
2.
3.
4.

Add 0.4 g uranylacetate in 10 ml distilled water

Stir at room temperature in the dark till dissolved
Store at 4C in the dark
Filter through a Millipore filter before use

74

12.17.

Suppliers

SPI: http://www.2spi.com
Copper or nickel grids (Hexagonal, 100 mesh):Stork-Veco Zoetermeer, NL
Site :
http://www.spgveco.com/precision+metal/technologies/electroforming?produc
t_id=146
Decon: http://www.decon.co.uk
Diamond knives: Diatome, Bienne, Switserland.
Site: http://www.diatome.ch/.
Gelatin: Rousselot 250 LP30.
Site: http://www.rousselot.com/
Microtomes etc : Leica Vienna Austria.
Site : http://www.leica-microsystems.com/products/electron-microscopesample-preparation/

75

13.

Addendum

13.1.

Protein A- G- L affinities

SpecieS
Human

Mouse

Rat

Cow

Goat

Sheep

Horse

Rabbit
Guinea Pig
Pig
Dog
Cat
Chicken
Legend:

Antibody ClasS
Total IgG
IgG1
IgG2
IgG3
IgG4
IgM
IgD
IgA
Fab
ScFv
Total IgG
IgM
IgG1
IgG2a
IgG2b
IgG3
Total IgG
IgG1
IgG2a
IgG2b
IgG2c
Total IgG
IgG1
IgG2
Total IgG
IgG1
IgG2
Total IgG
IgG1
IgG2
Total IgG
IgG(ab)
IgG(c)
IgG(T)
Total IgG
Total IgG
Total IgG
Total IgG
Total IgG
Total IgY

Protein A
S
S
S
W
S
W
NB
W
W
W
S
NB
W
S
S
S
W
W
NB
NB
S
W
W
S
W
W
S
W
W
S
W
W
W
NB
S
S
S
S
S
NB

Protein G
S
S
S
S
S
NB
NB
NB
W
NB
S
NB
M
S
S
S
M
M
S
W
S
S
S
S
S
S
S
S
S
S
S
NB
NB
S
S
W
W
W
W
NB

Protein A/G
S
S
S
S
S
W
NB
W
W
W
S
NB
M
S
S
S
M
M
S
W
S
S
S
S
S
S
S
S
S
S
S
W
W
S
S
S
S
S
S
NB

Protein L
S*
S*
S*
S*
S*
S*
S*
S*
S*
S*
S*
S*
S*
S*
S*
S*
S*
S*
S*
S*
S*
NB
NB
NB
NB
NB
NB
NB
NB
NB
?
?
?
?
W*
?
S*
?
?
NB

W= Weak
binding
M= mediuMbinding
NB= no binding
S= strong binding
? = information not available
*= Binding to Protein L will occur only if the immunoglobulin has the
appropriate kappa light chains.

Source: http://WWW.piercenet.com/files/TR0034-Ab-binding-proteins.pdf

76

13.2.

Rousselot gelatin

77

13.3.

Short incubation schedule.

Labeling with protein A-gold, the mentioned times are just guidelines. Shorter incubation
times may decrease the amount of labelling. The incubations are performed at room
temperature unless stated otherwise.
In our standard immunolabeling procedure, thawed cryosections on carbon-coated
Formvar copper grids are floated section side down according to the next protocol:

1.

Put the grids on 2% gelatin under a lamp

2.

Put grids on 2% gelatin at 40C in a stove

3.

Rinse with PBS + 0.1% glycine

4.

Block with PBS/BSA 1.0%

5.

Incubate on 5 l specific antibody (AB) diluted in PBS/BSA 1.0% .

6.

Rinse with PBS

7.

Protein A-Gold in PBS/BSA 1.0% 5-10 l drops 2

8.

Rinse with PBS

9.

Stabilize the reaction on 1% glutaraldehyde in PBS

+/- 5 min
20 min
4/5 x 1 min
3 min
20-60 min
4 x 2 min
0 min
4 x2 min

10. Rinse with distilled water fresh, not from plastic bench-bottles

5 min
10 x 1 min

11. Incubate with Uranyloxalicacetate PH7

5 min

12. Rinse once briefly with distilled water

13. Rinse once with ice cold MC-UA (big drop).
14. Incubate on a fresh drop of ice cold MC-UA

5 min.

15. Loop out the grids and remove the excess of MC-UA with filter paper
16. Air dry
Never let the grid become wet on the back surface or dry out during the incubation
procedure, this will generate many background gold particles.

78

14.

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81