FROZEN SECTION PROCEDURE

1. Obtain frozen tissue, preferably frozen in liquid nitrogen. It is imperative that the tissue be frozen as quickly as possible in order to avoid ice crystal formation resulting in artifact and poor morphological preservation.

2. Make sure the cryostat is at proper operating temperature -20 C to -30 C. Place a small amount of OCT or other suitable frozen section embedding medium on a cryostat object disk (make sure the disk is at room temp. before mounting the specimen).

3. Position the frozen specimen in the center of the object disk and place the disk on the cryobar in the cryostat to begin the quick freeze process.

4. Using "Histo-Freeze" or other appropriate aerosol refrigerant, spray around the periphery of the object disk, as the OCT freezes it will begin to turn from a clear gel to white solid substance. Proceed quickly to step # 5.

5. Before the disk is frozen solid add enough OCT to cover the top the specimen and quickly place a heat extractor on top of the specimen. The heat extractor serves two purposes, (1) rapidly freezes the OCT and tissue and (2) produces a flat embedded surface for easy cutting.

6. Spray with refrigerant if necessary to expedite the freezing process.

7. Place the object disk in the microtome object disk holder and tighten the set screw or clamp.

8. Make sure that there is enough clearance between the block and the microtome knife.

9. Move the block toward the knife edge. Make sure the ratchet is disengaged from the micrometer gear. Turn the flywheel with the right hand and begin turning the gross adjust wheel (on the down stoke) slowly with the left hand. Face off enough OCT until a full section of the specimen is visible.

10. Engage the ratchet on the micrometer gear, cut and discard the first two or three sections.

 

11. Have the proper fixative (95% ETOH for H&E) and slides ready. Turn the flywheel with the right hand. As the block comes in contact with the knife edge the section will move down the blade and begin to curl. Hold the section down with as little force as possible and guide in along the blade using a camel hair paint brush in the left hand. Continue the cut until a full specimen section has been obtained, but stop before passing through the remaining OCT. One edge of the section is held flat with the paint bush and the other with the knife edge.

12. Pick up the slide with the right hand and turn it so that the top side is facing toward the knife blade.

13. Carefully lower the slide onto the blade, keeping the slide parallel to the section. As the tissue comes into contact with the slide the OCT and tissue will melt causing the tissue to adhere to the slide.

14. Place the slide in fixative. If staining H&E sections, use 95% ETOH and fix for 30 sec.