Explain the stepes of preparation samples with tutorial video

1. The principle of frozen sections
Presented by
Dr. Hadeel khalaf
Alboaklah
Dr.
MuntaderMohammed
Kani

2. THEORETICAL CONSIDERATIONS
 The principle of cutting frozen sections is
simple: when the tissue is frozen, the interstitial water in the tissue turns to ice,
and in this state the tissue is firm with the
ice acting as the embedding medium.
 The consistency of the frozen block may be altered by varying the temperature
of the tissue. Reducing the temperature will produce a harder block; raising the
temperature makes the tissue softer.
 The majority of non-fatty unfixed tissues section well at −25°C. The sectioning
of
fixed tissue requires a block temperature of approximately −10°C or warmer

3. PREPARING TISSUE FOR FREEZING
 Tissue for freezing should be frozen or fixed as promptly as possible after
cessation of circulation to avoid morphological distortions and damage
due to
 Tissue drying artifact.
Autolysis - The destruction of tissues or cells by the action of substances,
such as enzymes, that are produced within the organism. Also called self-
digestion

4. FREEZE DRYING TECHNIQUE
 Little used in routine
• Used in immunohistochemistry
•The technique minimizes the:
•Loss of soluble substances
 Displacement of cell constituents
•Chemical changes of reactive group

5. Principle
• 1-Rapid freezing of fresh tissue -160C
(quenching) then removal of water in form of ice by sublimation at high
temperature -40 C
 The freeze dried blocks are then raised to room temperature and either
fixed by vapor 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

6. APPLICATION OF FREEZE DRYING
 Demonstration of hydrolytic enzyme
 Fluorescent Antibody studies
 Mucosubstance
 Proteins
 Scanning EM

7. CRYOSTAT
 Cryostat is a device by which temperature can be maintained in a low level.
In pathology and histology it is known as chamber containing a microtome
for sectioning frozen tissue
 Electronic temperature control.
 The temperature can be depending on the tissue being cut usually
between -20 to -30 C

8. OPTIMAL CRYOSTAT CUTTING
TEMPERATURES FOR UNFIXED
TISSUES
 Brain, lymph node, liver, kidney, spleen And testis at -12 to -16 C.
•Breast, skin,thyroid,adrenal,muscles and prostate at -18 to -30 C.
 Soft tissues cut better at slow rate while the hard tissues better at slightly
faster rate.
 -Fixed tissues is more difficult to cut well.
 Cutting section pick up on slides or cover-slips,the 40C difference in
temperature between section & slide enough to adhere the section to
slide.
 Fixed section have a tendency to detach during staining , to avoid this coat
the slide in gelatine-formaldehyde mixture, or poly-l-lysine.

9. Preparation and Staining of Frozen
Tissue Sections
 For use with Pharmingen reagents
 Materials need:
 2-methylbutane (isopentane)
 Liquid Nitrogen
 Dry ice
 Peel-Away® base molds
 Frozen tissue matrix (OCT® or Cryomatrix®)
 Long forceps
 Necropsy tools
 Superfrost Plus slides

10. Procedure
 Label base mold and partially fill the mold with frozen tissue matrix.
 Sacrifice animal by prescribed and approved euthanasia techniques.
 Remove desired tissues, trim and cut tissue no more than 5 mm thick. Place in pre-
labeled base molds filled with frozen tissue matrix. Arrange tissue in the matrix near the
bottom so tissue is easily exposed when sections are cut.
 Place a stainless steel beaker of 2-methylbutane in liquid nitrogen and allow to cool
adequately. Place base mold with tissue into the beaker of cold 2-methylbutane and
quickly immerse the block. Allow the tissue matrix to solidify completely and remove
block from 2-methylbutane and place on dry ice or in the -20°C cryostat. NOTE: If block
is left in 2-methylbutane too long, the block may crack.
 Store blocks in the -80°C freezer until ready for sectioning.

11. Sectioning of Frozen Tissues
 Before cutting sections, allow the temperature of the block to equilibrate to
the temperature of the cryostat (typically -20°C).
 Place the tissue block on the cryostat specimen disk. Adjust the positioning of
the block to align the block with the knife blade. Cut tissue block until the
desired tissue is exposed.
 Cut sections of the desired thickness (usually 5 µm), place the sections on a
Fisher Superfrost slide and dry overnight at room temperature (RT).
 Fix slides by immersion in cold acetone (-20°C) for 2 minutes or other suitable
fixative (e.g. alcohol, formal alcohol, formalin, etc.), air dry at RT and proceed to
staining.
 Alternatively, the frozen section slides can be stored for a short period of time
at -70°C in a sealed slide box. When ready to stain, remove slides from freezer
and warm to -20°C in the cryostat or -20°C freezer, fix for 2 minutes in cold
fixative (acetone or other suitable fixative) and allow to come to RT to continue
with the staining.

12. Tissue Processing Steps for Frozen
Samples