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Frozen sections are commonly used for:

1. Rapid pathologic diagnosis during surgery
2. Diagnostic and research enzyme histochemistry
3. Diagnostic and research demonstration of
soluble substances such as lipid and
4. Immunoflourescent and immunocytochemical
5. Some specialized silver stains, particularly in

2 methods of preparing
frozen sections:
1. Cold knife procedure
2. Cryostat procedure (cold

Cold knife procedure



A piece of filter paper soaked in gum syrup is placed on the

microtome stage, and short bursts of CO2 are applied, freezing
the filter paper to the stage.
The selected block of tissue, approximately 3-5 mm thick is
oriented in the stage, applied with a few drops of gum syrup and
frozen solid with several intermittent bursts of CO2 each for 1-2
seconds duration at intervals of around 5 seconds. It should be
frozen just to the point where it will be firm enough to section
Then lift the tissue up to the knife manually and trimmed until the
surface is flat
The surface is then warmed with the finger until the hard frozen
tissue starts to thaw and becomes visible to the naked eye. This is
the Dew line, the point at which sections may be cut at 10 micra

5. Sections do not form ribbons but rather stick to the

knife blade and should be removed with a camel hair
brush or finger moistened with water.
6. Transfer these to a dish of distilled water to separate
and picked up individually for mounting and staining
7. Using a cold knife in a controlled cold environment,
optimum condition for sectioning shall be provided by
the following temperatures:
knife- 40 to -60C
tissue 5 to -10C
environment- 0 to -10C

Cryostat procedure
This method makes use of a cryostat, an apparatus
used in fresh tissue microtomy, consisting of an
insulated microtome housed in an electrically driven
refrigerated chamber maintained at temperature near
-20C where microtome knife, specimen and
atmosphere are kept at the same temperature. The
optimum working temperature of cryostat is -18 to
The tissue for freezing should be fresh, and freezing
should be done as quickly as possible. Slow freezing
can cause distortion of tissue due to ice crystal artifacts.

methods of freezing
Liquid nitrogen
Isopentane cooled by liquid
Carbon dioxide gas
Aerosol sprays

Liquid nitrogen
use in histochemistry &
procedures and is the most
rapid of the commonly
available freezing agents.

A pyrex glass beaker is usually
suspended in a flask of liquid nitrogen
until half-liquid and half-solid stage is
reached. The beaker is removed from
the liquid nitrogen when small
crystals start forming on the side of
the beaker (approx. -170C) and the
tissue to be frozen is dropped into the
cooled liquid nitrogen

Aerosol sprays
has become popular in recent years
and is adequate for freezing small
pieces of tissues except muscle
The success of fresh tissue sectioning
depends on the large extent on the
temperature both of the tissue and the

Care of the Cryostat

cryostat should be left on at all times. It takes at least one
hour for a knife to come down to operating temp, so that a
spare knife should always be kept inside the cryostat cabinet
soft tissue paper, either dry or moistened with absolute
alcohol, may be used for cleaning the knife and anti-roll plate.
the cryostat should be defrosted during the weekend,
including cleaning and oiling of microtome with special lowtemperature oil

Special Processing Techniques

These techniques are

important in histochemical
evaluation particularly in the
preservation of ENZYMES .

Freeze drying
a special way of preserving tissues by
rapid freezing (quenching) of the fresh
tissue at (-160C) and subsequently
(desiccation) by a physical process of
transferring the still frozen tissue block
in a vacuum at a higher temp, e.g.
(sublimation) without the use of any
chemical fixative.

1. Tissue (around 2 mm. thick) is plugged into
isopentane or propane-isopentane chilled to -160 to
-180 C with liquid nitrogen
2. Tissue is solidified within 2-3 seconds, (prevents
formation of ice crystal artifacts, autolysis and
3. Transferred to a high vacuum drying apparatus(-30 to
-40 C) depending on the size of the tissue. Water is
sublimated and dehydrated from the tissue within 2448 hours (desication is completed)
4. Tissue is removed from the apparatus and embedded
in molten paraffin wax, water soluble wax or celloidin.
5. Infiltration or impregnation in a vacuum embedding
6. Sectioning ( routine procedure)
7. Staining ( depending on the component desired)

1. Produce minimum tissue shrinkage
2. Tissues are processed in fresh state
3. Minimal chemical changes in the cell esp. the
4. Less displacement of tissue and cell
5. Very important for enzyme studies


Time consuming
Tissues are generally more difficult to section
Tissues are brittle and inadequately supported
due to relatively short period of wax impregnation
5. Not recommended for routine purposes

In addition to demonstrating hydrolytic enzymes,

mucous substances, glycogen and proteins,
freeze drying may be used for special studies,
1. Immunocytochemistry
2. Fluorescent antibody studies of polypeptide and
polypeptide hormones
3. Autoradiography
4. Microsoectroflourimetry of autoflourescent substances
5. formaldehyde-induced flourescence of biogenic amines
( to demonstrate 5-hydroxytryptamine, adrenaline,
nonadrenaline and other catecholamines
6. Scanning electron microscopy

Freeze substitution
similar to freeze drying except that it involves rapid
freezing and subsequent infiltration and embedding
frozen tissue instead of being subjected to
dehydration in an expensive vacuum drying
apparatus , is fixed in Rossmans fluid or Osmium
tetroxide in 1% acetone for 1 to 6 days at a
temperature of -60 to -70C respectively. Infiltration
and embedding is carried out in the same way as in
paraffin sections
this is more economical than freeze-drying and is
recommended for routine purposes

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