Microtome
Open valve to CO2 tank and cool stage till frozen.
Cut straight across the cerebellum so the brain can stay upright.
Apply 1% ethanol to frozen stage as the base.
when the ethanol is halfway frozen, add a layer of 30% sucrose to the base.
Once the sucrose is halfway frozen, place the brain dorsal side towards the blade.
Add more sucrose around the tissue and allow the whole thing to freeze.
Adjust the stage so the tissue is below the blade.
place the blade in the designated spot and tighten screws to secure the blade.
Obtain a brush to handle the tissue and sectioned dish with distilled water to place the tissue.
Start cutting tissue into the desired thickness and fully extend cutting arm for automatic adjustments.
Once done cutting, remove blade, wash with soap and water and then dry. Coat the blade in oil and store until next use.
To mount, place one section in a dish of water. Let it float to the top and place the slide under tissue in the water.
Use a brush to maneuver the tissue on to its spot on the slide.
Allow tissue to dry on the slide before staining.
Nissl Staining
Obtain six containers: one with distilled water, one with 70% alcohol, one container with absolute alcohol, the fourth container with clear safe, the fifth container with the cresyl violet stain.
Glass side facing outwards, place each slide into the designated spot in the distilled water container. Leave for 2 minutes.
Take the slides and transfer them to the 70% alcohol container, leave for 2-4 minutes (closer to 2).
Take the slides and transfer them to the container with the absolute alcohol and let them sit for 5 minutes.
Then move the slides to the container with the clear safe. Any smoke means you have to go back to step 4. Leave in clear safe for 5 minutes.
Move the slides back to the absolute alcohol for 4 minutes, then the 70% alcohol for 4 minutes, and the water for 2 minutes.
Then put the slides in the stain for 20 minutes, put EtOH in the last container, and place the container after the water.
After 2o minutes, move the slides to the water and discard the colored water, then refill it with more distilled water.
Let the slides sit in the water for 2 minutes and transfer it to the acid alcohol.
With the acid alcohol, discard the pink-colored solution and add more of the acid alcohol.
Move the slides to the 70% alcohol then the absolute alcohol and finally to the clear safe.
To prepare the cover slips, lay them out.
Open the paramount and using a Q-Tip place it on the bottom edge of the stained slide (on the brain side).
Take the cover slip placing it perpendicular and lay it flat to the slide and gently tap it to make sure the edges are even.
Let the slides dry.