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Methods to study Histology

Krishna T
Histology
Cell
Tissue
Organ
Organ system
Homeostasis
How to get the histology slides?
How to get tissues for study
Steps in tissue preparation
Fresh tissues from the body
1. fixation
Formalin ( 10% formaldehyde)
Osmium tetroxide for EM
Mechanism - Forms cross links with proteins (Lysine)
2. Embedding gives support for tissue slicing
Paraffin or plastic resin
3. Washing & dehydration (dehydration by graded alcohols in
ascending order)
4. clearing to remove paraffin & alcohol
By xylol or tulol
5. block making
How to get the histology slides?
6. section cutting 5-10 thick sections with microtome
7. mounting on glass slide ( adhesive albumin)
8. clearing xylol / tulol
9. rehydrate alcohols in descending order
Staining
nuclear stain Hematoxylin ( basic stain & water soluble)
counter stain Eosin ( less water soluble but soluble in
alcohol) dehydrate in ascending order
10. Clearing xylol / tulol
11.Mounting medium cover glass
Special situations
Staining routine stain H&E
Some structures are seen/ preserved (large molecules like nucleoproteins,
cytoskeleton proteins, ECM proteins- collagen, membrane proteins)
some are not seen/lost (small molecules -t-RNA, large molecules like
glycogen & Proteioglycans are dissolved, )during the fixation/staining process
Special fixatives to retain membrane ( phospholipids)
Permanganate & osmium for EM
For Elastic fibers Orcein/ Resorcin Fuscin
For reticular fibers Silver impregnation
Histochemistry & Cytochemistry

Specific binding of dye with particular molecule


Fluorescent dye labeled antibody to cell component
Enzyme activity
Autoradiography radio isotopes tagged with precursors of a

molecule molecule incorporated into cell/ tissue before fixation


Basis of staining

ACIDIC DYES BASIC DYES


Eosin Hematoxylin /Methylene
blue

Carry net negative charge Carry net positive charge


React/bind with cationic With anionic components
components of the of cell/tissue
cell/tissue
Less specific (as Highly pH specific
compared with basic
dyes)
Acidophilic / Eosinophilic Basophilic substances
(cytoplasmic filaments, ( Po4 of Nucleic acids,
intracellular membranous So4 of MPS, CO proteins)
What is special about Hematoxylin?

Mostly resembles basic dye but it is a


mordant (helps to form links between tissue
fragment & the dye)
It will not dissociate in sequential staining

process unlike other basic dyes


Metachomasia
What is it ? Absorb certain wavelength
of light and emit different wavelength
Why Metachomasia ? Polyanions of
tissues bind with dye molecules result in
polymer or dimers of dye molecules
appear as different color rather than
expected ( methylene blue gives red or
purple color)
What are metachromatic substances?
Ionized So4, Po4 of cartilage
Where you find it? Mast cell granules
(heparin) & rER of Plasma cells
PAS =Periodic Acid Schiff

Special stain
PAS positive substances
Carbohydrate (glycogen) or
carbohydrate rich molecules,
Basement membrane, reticular fibers
Periodic acid cleaves bond between
carbon atoms form aldehyde
group
Aldehyde binds with Schiff to
produce magenta or pink color
Feulgen stain for Nuclear Proteins

Acid hydrolyses or cleaves proteins from


deoxyribose of DNA leads to opening of sugar
group & formation of aldehyde
Schiff binds and gives magenta color to
aldehyde
Can be useful to quantify amount of DNA ( by
using spectrophotmetry of Feulgen stained
tissue)

Why RNA cannot be stained by Feulgen?


Enzymatic digestion
For the confirmation of specific substances
Pretreatment of sections with specific

enzymes
Diastase/amylase for glycogen
DNA ase for DNA
Enzyme Histochemistry
Localization of enzymatic activity in tissues
Best fixation mild aldehyde ( formalin)
Basis localized reaction production of

enzyme activity
Used for acid & alkaline phosphatase, ATP
enzyme
ases
AB (substrate) + T (trap) AT ( reaction
product) + B (Hydrolyzed component of
substrate)
Immuno Histo Chemistry (IHC)
Antibody ( Immunoglobulin) conjugated with
fluorescent dye( most common is Fluorescein)
+ Antigen ( foreign protein)
Fluorescein absorbs UV light and emits
green fluorescence can be seen under
Fluorescent microscope (IF- Immuno
Fluorescence)
Example :- actin (Antigen) of Rat infected
to Rabbit blood of Rabbit ( have poly -
clonal antibodies for Rats actin/ anti rat actin
antibodies) bind with Fluorescent dye
Monoclonal Antibodies

Specific antigen Multiple Myeloma pts.


(actin of rat)

B lymphocytes of
Immunized rabbit
Monoclonal B ells

Hybridoma cells

Single specific type of antibodies
(Monoclonal)
( against Actin)
Clinical Significance of Monoclonal
Antibodies

Diagnosis of tumors(tumor markers) &


Infections( HIV, Infectious Mononucleosis)
Classify sub types (B -cell and T- cell

lymphomas)
Treatment Anti-TNF- antibodies in

inflammatory disorders
Immunological Methods
Immuno -fluorescence
Direct (one step, less sensitive)
&
Indirect ( more sensitive, Expensive, labor intensive,
cant easily run in automated) methods
Immunoperoxidase method
Enzyme is used ( horse raddish peroxidase) to color
colorless substrate into colored insoluble product
Other Methods

Hybridization: for localizing


mRNA/DNA (NA)
In Situ Hybridization: Binding
( Probe + NA) in cell/tissue
FISH: If Fluorochrome is used
in Hybridization technique
Autoradiography: by tagging
the precursor molecules
(Amino acids) followed by
synthesis of large molecules
(NA) localize the particular
tagged molecule
Microscopy
Resolution/ Resolving power (RP): the
distance by which two objects must be
separated to be seen as two objects
RP of
Unaided Human retina : 0.2 mm
Light Microscope (LM) : 0.2
Electro Microscope (EM) : 1.0 nm
LM: we see only two dimensional pictures,
orientation of cut gives different patterns
Artifacts: error in preparation process
orientation of cut
Three dimensional picture

How you get it?


Types & Advantages of Microscopes

1. Phase contrast M:
can see live (unstained) tissue
Light passing thru denser tissue of higher
refractory index out of phase from the rest
look darker
Uses : identify cells in tissue cultures
Modification: Interference M: quantification of
tissue masses helps in study of surface properties
of cells

What happens to the tissues during routine staining


process?
Types & Advantages of
Microscopes
2. dark Field M: special condenser illuminates
specimen with strong oblique light
Uses:
In auto radiography
Study crystals in urine
Study microbes- slender spirochetes ( *Treponema pallidum)
3. Fluorescent M: emits light in visible range when
exposed to UV light
Technique: filters are used between light source & specimen
Naturally fluorescent substances: Vitamin A, Neuro-
transmitters
Uses
Tracing pathways of nerve fibers,
To detect growth markers of mineralized tissues

*What is the disease caused by this bug?


Types & Advantages of Microscopes

4. Confocal scanning M:
Conjugate with focal point of lens
Computer software reconstitutes the image from the data
Major difference from LM: addition of detector aperture
(pin hole)
Uses: can see 3D pictures
5. ultra violet M:
Depends on absorption of UVL by specimen
Results are recorded photographically (cant be seen
directly why?)
Uses
Study of nitrogen bases ( in NA)
Study amount of DNA/RNA in cells *Clinically helps in study
of ploidy in tumors

Highly aneuploid tumor What is its Significance ?


Types & Advantages of Microscopes

6. Polarizing M: only difference is polarizer


(polarizing filter)
Birefringence: ability of crystalline or Para -
crystalline material to rotate the phase of polarized
light (double refraction)
Skeletal muscle & Leydig cells
*Amyloid protein: apple green
Uric acid: negative
Ca++ pyrophosphate

* ,, clinical Significance ?
Types & Advantages of Microscopes

7. Electron M (EM): specimen is in vacuum


Types: Transmission (TEM), scanning (SEM)
Mechanism: similar to LM except that beam of
electrons replace light source
Recording: photoelectric plate or video detector
Specimen preparation:
Fixation: Glutaraldehyde (cross links with proteins), Osmium
tetroxide (reacts with *phospholipids) makes cell/tissue
electron dense for image enhancement
Other steps are same as routine tissue processing except
Plastic is used for embedding
Diamond knives are used in microtome ( not metal knives)
To study membranes Freeze fracture technique {-
160C with glycerol (to prevent ice crystal formation)}
* Where you find ?
why diamond knives are used in EM?
Types & Advantages of Microscopes

7. Scanning (SEM)
It differs from TEM that electron beam
passes across the surface of spectrum
(not thru specimen as in TEM)
Resembles Television
Can see 3D pictures
8. Atomic Force M: most powerful
tool to study surface topography
Non optical M: works like finger tip
Has highest resolution power 50 pm
*Specimen need not be in vacuum

*what is the additional advantage?

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