Tissue Sectioning Procedure
Open valve to CO2 tank & cool stage until completely frozen.
Cut straight across cerebellum so brain stays level/upright.
Put 1% ethanol to the frozen stage, as a base layer.
When ethanol is halfway frozen, add a layer of 30% sucrose to the base.
When sucrose is halfway frozen, put brain on stage so the dorsal side faces towards the blade.
Add more sucrose around the tissue, & allow it to freeze.
Manually lower/raise the stage so the tissue is below where the blade will run.
Place blade in designated space. Tighten screws to hold blade in place.
Get a brush to handle the tissue & sectioned dish full of distilled water to place tissue.
Cut tissue into sections of desired thickness (50 microns) & fully extend cutting arm for automatic adjustments.
When finished sectioning the tissue, remove the blade, wash it w soap & water, & dry it. Coat in oil & store until next use.
For mounting, place one section in dish of water, float to the top & 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
Get 6 containers, placing the 6th in the back. Add distilled water to the first container.
Add 70% alcohol to the second container.
Add absolute alcohol to the third container.
Add clear safe to the fourth container.
Add cressyl violet stain to the fifth container.
Ensure glass side of slide is facing outwards; place into first container full of water. Put as many slides as there are spaces for, for about 2 minutes.
Take slides out of water & place into 70% alcohol (this dehydrates the tissue). Leave in alcohol for 2-4 minutes.
Take slides out of 70% and place into absolute alcohol. Leave in alcohol for 5 minutes.
Take slides out of absolute alcohol and place in the clear safe. If any smoke is visible, put slide back into the absolute alcohol. When there is no smoke, leave slides in the clear safe for about 5 minutes.
Repeat steps 6-9 but in the reverse order. 4 mins in alcohols, 2 mins in water. After 2 mins in water, slides can go in the cressyl violet.
Leave slides in stain for 20 mins.
Add acid alcohol to the sixth container, & place the container in between the containers with water & 70% alcohol
Put slides in water. When water changes color, replace w new water.
Put slides in acid alcohol. When it changes color (pink), replace w new acid alcohol. Must be done quickly
Put slides in 70% alcohol for as long as necessary
Put slides in absolute alcohol
Put slides in clear safe. Tissue should not be losing stain at this point.
Separate cover slips from the slides, & lay them out so they can be grabbed easily.
Apply permount to bottom edge of slides, making sure the slides are facing away from you. Put cover slip back on the slide carefully.