Open CO2 tank valve, and cool stage until completely frozen.
Cut straight across cerebellum so brain can stay level and upright.
Apply 1% ethanol to frozen stage as the base.
When EtOH is halfway frozen, add a layer of 30% sucrose to the base.
When sucrose is halfway frozen, place brain so that the dorsal side will face towards the blade.
Add more sucrose around the tissue and allow to freeze.
Manually lower or raise the stage so that the tissue is below where the blade will run.
Place blade in its designated space and tighten screws to hold the blade in place.
Obtain a brush to handle the tissue and sectioned dish full of distilled H2O to place tissue.
Begin cutting tissue into sections of the desired thickness (50 microns) and fully extend cutting arm for automatic adjustments.
When done cutting, remove blade, wash with soap and water, then dry. Coat blade in oil and store until next use.
For mounting, place one section in dish of water, float to the top and place slide under tissue in water.
Using a brush, guide the tissue to its spot on the slide.
When done mounting, allow your tissue to dry before staining.
Obtain 5 containers and fill with the following substances:
distilled water
70% alcohol
absolute alcohol
Clear Safe
cresylviolet stain
Take slides, and making sure that the glass side of the slide is facing outwards, place each slide into the first container with the distilled water. Leave the slides in for 2 approx. 2 minutes.
Take the slides out of the distilled water, and place them in the 70% alcohol. Leave them in for 2-4 minutes.
Move the slides from the 70% alcohol to the absolute alcohol. Leave them in for about 5 minutes.
Move the slides into the Clear Safe. If you see something similar to smoke it means that you haven't fully dehydrated your tissue and you need to put it back into the alcohol. Leave the slides in for approx. 5 minutes.
Move the slides from the Clear Safe back into the absolute alcohol, and leave them in for approx. 4 minutes.
Move them from the absolute alcohol into the 70% alcohol for 4 minutes.
Move them into the distilled water for 2 minutes.
Then, place the slides into the stain.
Leave the slides in for 10-20 minutes.
Adding acid alcohol to the 6th container.
Once the acid is acid, place the acid between the water and the 70% alcohol.
Make sure waste container is nearby. Move slides out of stain and into water. When water starts to change color, dump it out and refill it.
Move it into the acid alcohol. When you see a color change, dump it out and repeat the same process as the water.
Move into the 70% alcohol. Leave it in for as long as needed.
Move into the absolute alcohol, and then the Clear Safe.
Prepare coverslips for slides.
Remove a slide from the Clear Safe, hold it at an angle facing away from you. Take paramount and use a stick to apply it to the edge facing away from you.
Take a coverslip and place it perpendicular to the slide, halfway down the coverslip. Flatten it onto the slide. Tap it on each side to make sure it's even.
Repeat for the remaining slides.
Leave the slides on a flat surface to dry.