Frozen section /orthodontic courses by Indian dental academy

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H. & E. STAINING
IN FROZEN SECTION
INDIAN DENTAL ACADEMY
Leader in continuing Dental Education
www.indiandentalacademy.com

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References :-
• Bancroft J. D.,Stevens A., Turner R. D. ,Museum and other
demonstration techniques, In Theory and Practice of Histological
techniques, fourth edition, Churchill Livingstone. page no.699-712.
• Culling C. F. A., Allison R. T. ,Museum Techniques, In Cellular
Pathology Techniques, fourth edition, Butterworth. page no-523-540.
• Drury R. A. B. ,Wallington E. A. Museum Techniques and injection
methods, In Carleton's Histological Techiniques,fourth edition, Oxford
University Press. page no-390-401.

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Contents
3. Microtomy
a. CO2 freezing microtome
b. Thermoelectric cooling devices
c. The Cryostat
d. Aerosol sprays
1. Preparation of the frozen sections
2. Preparation of the material

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4. Rapid Frozen Section Technique
• Synthetic resin embedded tissue
• Freeze-dried Tissue.
• Freeze Substitution

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Introduction
Frozen Section Uses :-
1. Rapid production of the sections for urgent diagnosis.
2. Use in diagnostic and research enzyme histochemistry
where enzymes are labile.
3. Use in immunofluorescent method

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4. Use in immunocytochemical method.
5. Diagnostic & research non-enzyme histochemistry
e. g.lipids and some carbohydrates.
6. Some silver methods, particularly in neuropathology

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∙ When tissue is frozen the water within the tissue turns to be
ice and in this state tissue is firm, the ice acting as an
embedding medium.
Frozen Section
Theoretical Consideration :-

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∙ The consistency of the frozen section block can be altered by
varying the temperature of the tissue.
∙ The sectioning of the fixed tissue require a block
temperature of the about minus 10 or warmer.
Frozen Section

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• It is a refrigerated cabinet in which a modified microtome is
housed.
• All controls to the microtome are operated from outside the
cabinet.
• First microtome was introduced in the year 1954; at present
rotary microtome is the type of choice.
The Cryostat

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Various advancements in the design of the microtome are as :
•Electronic temperature control.
• Electronically controlled advancement and retreat of the block.
• Specimen orientation facility.
• Digital demonstration of the cryostat temperature and section
thickness.
• Mechanized cutting speed facility.

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The best quality cryostat sections are produced from fresh
unfixed tissue, which has been rapidly frozen by one of the
techniques outlined below. The conditions in the cryostat
itself must be optimal -
Block temperature correct for tissue being cut.
Microtome operating correctly.
Anti - roll plate adjustment correct.
Cryostat Technique

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Methods of freezing are -
Liquefied Nitrogen : - 190 0
C
Isopentane cooled by liquid nitrogen : - 150 0
C
Carbon dioxide ‘Cardice’ : - 70 0
C
Aerosol sprays : - 50 0
C
Freezing of the fresh unfixed tissue
Tissue for freezing should be fresh, and freezing should be as
rapid as possible.

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For almost all diagnostic purposes in routin laboratory
cryostsat sections of unfixed tissues are suitable, although
certain methods require post fixation in cold formal-calcium.
Fresh Tissue & The Cryostat

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Tissue prepared in such a way is fixed under controlled
conditions. The tissue must be absolutely fesh and placed in
formal–calcium solution at 40
C. The tissue is fixed at this
temperature for 18 hours

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When the tissue block is ready for sectioning ,the tempt of
the microtome & cryostat chamber should be checked.
Most unfixed material will section well between -15to -23 C.
Cryostat Sectioning

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Unfixed tissues containing moderate to large amount of fat
will require as cold a temperature as the cryostat is capable of
producing.

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Most fixed tissues will section best within the range – 7 0
C to
-120
C depending upon the hardness of the tissue.
Cabinet temperature
Microtome
Blade or Knife
Anti-roll plate
Sectioning technique
Soft tissue cut better at a slower speed and hard tissue cut at
slightly higher speed. Fixed tissue is more difficult to cut well.www.indiandentalacademy.com

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Microtome Set-up

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Microtome

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Cryostat

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Freezing Tissue In The Histobath

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Processing

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Sectioning

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Tissue Sectioning

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Tissue Sectioning

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Tissue Sectioning

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Tissue Sectioning

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Frozen Section Stains

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Slides For Frozen Section

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Kidney Section Immediately Fixed

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Rapid Fixing In Broncho-alveolar Carcinoma

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It is a technique of rapid freezing of fresh tissue at -1600
C and
subsequent removal of water molecules (in the form of ice)
by sublimation in vacuum at a higher temperature e.g. – 400
C.
Freeze Drying

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The freeze dried blocks are then raised to room temperature
and either fixed by vapour fixative or embedded in a suitable
medium.
Freeze -drying can be considered in four stages:-
a. Quenching
b. Drying
c. Fixation & embedding
d. Subsequent treatment

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This instantly stops the chemical reaction and diffusion in
the tissue.
It brings the tissue into a solid state in which unbound tissue
is changed into small ice crystals, which are subsequently
removed in the drying phase.
Quenching

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This part of the technique is the most time consuming as
some tissue contains 70-80% water by weight and this has to
be removed without damaging the tissue.
Drying

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Drying can be divided into three distant stages -
1. Introduction of the heat to the tissue to cause sublimation of
the tissue.
2. The transfer of the water vapor from the ice crystals through
the dry part of the tissue.
3. The removal of the water vapor from the surface of the
specimen.

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When the tissue is completely dry, it is allowed to come to
room temperature.
This delicate tissue is ready for embedding and processing.
Vapor fixation -
Fixatives used for vapor fixation are formaldehyde,
glutaraldehyde and osmium tetra oxide.
The most common and excellent fixative is formaldehyde
because it gives excellent preservation of the tissue
component and tissue can be used for all histochemistry
with exception of the enzymes.
Fixation & Embedding

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Application of Freeze drying
• Demonstration of hydrolytic enzymes
• Fluorescent antibody studies
• Autoradiography
• Microspectrofluorimetry of autofluroscent
substances
• Formaldehyde-induced fluorescence
• Mucosubstances
• Proteins
• Scanning electron microscopy

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This technique originated by Simpson (1941) involves the
rapid freezing of small pieces of tissues in a similar manner
as for freeze drying.
Substitution of the ice in such tissue by placing them in
dehydrating agent at sub zero temperature.
The preservation obtained with the freeze substitution is
excellent. The technique produces considerable amount of
shrinkage.
Freeze Substitution

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Frozen section /orthodontic courses by Indian dental academy

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