Tissue sectioning procedure:
Open valve to the CO2 tank, and cool the stage until totally frozen
Cut straight across the cerebellum so the brain can stay level and upright
Apply 1% ethanol to the 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 the brain so the dorsal side faces toward the brain
Add more sucrose around the tissue and allow to freeze
Manually lower or raise stage so the tissue is below where the blade will run
Place the 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 H20 to place tissue
Begin cutting tissue into sections of the desired thickness (50 microns) and fully extended arm for automatic adjustments
When done cutting, remove blade, wash with soap and water, then dry. Coat blade in oil and store for 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 tissue to dry before staining
Nissl staining procedure:
Obtain six containers with the sixth placed in the back
Add distilled water to container #1
Add 70% ethanol to container #2
Add absolute alcohol to container #3
Add Clear Safe to container #4
Container #5 (placed in the front) will contain cresyl violet
Place each of the slides into the distilled water for two minutes
Place the slides in the 70% ethanol to dehydrate the tissue for four minutes
Place the slides in the absolute alcohol for 5 minutes
Move the slides to the clear safe (If there’s a smokey appearance than dehydration wasn’t achieved so it must go back in the absolute alcohol. Leave in for around 5 minutes or until the tissue is fully cleared
Again, place the slides in the absolute alcohol for 5 minutes
Again, place the slides in the 70% ethanol to dehydrate the tissue for four minutes
Again, Place each of the slides into the distilled water for two minutes
Place the slides into the cresyl violet stain Leave the slides in the stain for 20 minutes
Prepare container #6 with acid alcohol
Place container #6 in between the water and the 70% alcohol
Move the slides out of the stain and into the water (refill water)
Then move into the acid alcohol. Once there is a color change refill the acid alcohol
Then move the slides into the 70% alcohol (eye ball the color
Move the slides into the absolute alcohol container for several minutes
Then move the slides into the Clear safe container (you shouldn’t lose stain after this step)
Prepare the cover slips (one for each slide)
Apply paramount onto the slides with a q-tip
Apply paramount to the side of the slide facing away from you
Place coverslip perpendicular to the slide
Tap on each side to ensure they are properly aligned
Repeat for the remaining slides