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2 methods of preparing
frozen sections:
1. Cold knife procedure
2. Cryostat procedure (cold
microtome)
3.
4.
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
-20C.
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
nitrogen
Carbon dioxide gas
Aerosol sprays
Liquid nitrogen
use in histochemistry &
during
intra-operative
procedures and is the most
rapid of the commonly
available freezing agents.
Isopentane
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
knife
Freeze drying
a special way of preserving tissues by
rapid freezing (quenching) of the fresh
tissue at (-160C) and subsequently
removing
ice
water
molecules
(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.
Procedure
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
putrefaction)
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
oven
6. Sectioning ( routine procedure)
7. Staining ( depending on the component desired)
Advantages:
1. Produce minimum tissue shrinkage
2. Tissues are processed in fresh state
3. Minimal chemical changes in the cell esp. the
proteins
4. Less displacement of tissue and cell
constituents
5. Very important for enzyme studies
Disadvantages
1.
2.
3.
4.
Time consuming
Expensive
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
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