Slicing Brains on the Freezing Microtome

Tissue sectioning is extremely important for your histological analysis. It is therefore imperative that an instructor or a graduate assistant should closely supervise this procedure.

Materials

• Freezing Microtome and Stage
• Dry Ice and Scooper (for crushed dry ice)
• Small Paintbrushes

· Cell Wells

• Kim-Wipes
• Isopropyl Alcohol
• Hammer
• PB or PBS (depending on what stains are to be performed)
• Hand Towels
• Distilled Water
• Pipettes

· Scalpel or Razor

• Forceps
• Permanent Marking Pen
• Cryoprotectant (for tissues to be stored)
• PIKE OIL

Procedure

1. Place the stage in its holder and secure it. Be sure that the cutting arm will slide smoothly past it (if it won’t, lower the “stage holder”).

2. It is extremely important that the stage is leveled. Use a carpenter’s level to verify. If the stage is not leveled, loosen the screw holding the stage in place, adjust the position of the stage, tighten the screws and verify the positioning of the stage again).

3. Fill your cell wells using PB or PBS (depending on stains to be performed on the tissue sections as directed by the graduate assistant). Label the lids of the cell wells using a permanent marker as directed. Place the wells within your reach close to the freezing microtome.

4. Using the hammer, break a few pieces of the dry ice in chunks and place them in the wells on both sides of the stage using a hand towel. Dry ice can burn your hands, be careful handling it.

5. Carefully squirt isopropyl alcohol over the dry ice chunks without getting any on the stage itself. The reaction between the dry ice and the alcohol will produce vapors, which will freeze the stage. This takes a few minutes.

6. Slide the sectioning blade into its mount and secure it there. Drop the stage to its lowest point and fully retract the sectioning blade. The brain may only be mounted on the frozen stage ONLY after the stage has been dropped to its lowest point and the sectioning blade has been fully retracted.

7. Remove brain from solution (either sucrose or cryoprotectant). Using the scalpel, carefully notch one hemisphere (very superficial notch) from the front to back of the brain; this will serve as a guide so all sections are mounted with the same orientation later on slides.

8. Make a coronal cut through the cerebellum. The cut at this point needs to be made very carefully so that the base of the brain is now flat.

9. Once the stage has frosted over, carefully squirt distilled water in a filled square pattern on the frozen stage in the middle of what will be the path of the cutting blade. Stand the brain in an upright position on the freezing stage (where distilled water was squirted), with the cut end in the water on the frozen base. The ventral area of the brain should be facing the blade. Allow the water to freeze the brain into the frozen base, to prevent brain movement during sectioning.

10. Surround the entire brain with crushed dry ice. Allow the brain to reach optimal cutting temperature (at an optimal temperature brain has a whiter shade). After the brain has reached the optimal temperature, gently expose the cutting surface of the brain.

11. Make sure the frozen microtome “cutting setting” is set at the appropriate sectioning thickness. Adjust to the appropriate setting if necessary.

12. To begin sectioning, slide the blade across the brain in a moderately slow, controlled, yet firm fashion. The sections should ruffle up onto the blade.

13. Dampen a small paintbrush with PB (or PBS, depending which solution is being used), then gently glide the brush-head along the blade, brushing the section off the blade and onto the paintbrush. Carefully dip the brush-head in to the solution in the wells (PB or PBS), twisting the brush slightly as you pull it back out of the cell well, so that the section gently slides off the brush into the solution.

14. Once the section is removed from the blade, use a kim-wipe to remove moisture from both the upper and lower surfaces of the blade.

15. Once you have finished slicing the brain ask your supervisor whether the tissue should be stored in the refrigerator or cryoprotected and stored in the freezer.

WHEN DONE WITH USING THE FREEZING MICROTOME, CLEAN THE STAGE AND DRY IT, OIL THE EDGES OF THE SLIDING PART OF THE APPARATUS USING PIKE OIL AND COVER THE FREEZING MICROTOME.

CAUTION: Tissue sections are extremely fragile even when they appear intact. Be gentle in handling the brush and the tissue so you do not damage any sections. Handling the tissue in a rough manner may be damaging later (such as during staining or transferring tissue from wells) as it can weaken the tissue connections.

NOTE: You must also pay close attention to the condition of tissue as you are slicing the brain. The brain will “lack color” if it has become too hard (frozen) or may have visibly dark coloration if it is becoming too soft (thawed). It is therefore important that the temperature be monitored closely and adjusted to get consistently useful sections.