Staining Procedures

Tissue Sectioning

1.     Open the CO2 tank and completely freeze the stage

2.     Cut straight across the cerebellum

3.     Apply 1% Ethanol to the now frozen stage

4.     Add 30% sucrose once the Ethanol is halfway frozen

5.     Once sucrose is halfway frozen, place brain so that the dorsal side faces the blade

6.     Add additional sucrose around the tissue to freeze

7.     Move the stage so that the tissue is below the blade

8.     Correctly place the blade and tighten screws to hold it in place

9.     Obtain a brush and dish with distilled H2O

10.  Cut tissue into 50 micron sections and extend cutting arm

11.  After cutting, wash the blade with soap and water. Then coat blade in oil once dry

12.  To mount, place one section in dish of water and place the slide under the tissue in the water

13.  Use a brush to guide the tissue onto the slide

14.  Then, allow tissue to dry before staining.

Nissl Stain

1.     Obtain 6 containers and add distilled water to the first container, 70% alcohol to the second, absolute alcohol to the third, clear safe to the fourth, cresyl violet stain to the fifth 

2.     Take a slide and place it into the container with distilled water for 2 minutes

3.     Remove the slides from the distilled water and place them into the 70% alcohol for 4 minutes to dehydrate the tissue.

4.     Then move the slides to the container with absolute alcohol for 5 minutes

5.     Then move the slides to the clear safe for 5 minutes. If smoke appears, move the slides back to the container with absolute alcohol to properly dehydrate the tissue

6.     Then repeat steps 2-5 in the opposite order, and then place the slides in the stain for 20 minutes

7.     While the slides are still in the stain, add acid alcohol to the sixth container

8.     Then move the slides from the stain to the water and observe a color change. When this happens, discard the water and refill it

9.     Then move the slides to the acid alcohol and refill quickly once a color change takes place

10.  Move the slides to the 70% alcohol as long as necessary.

11.  Move the slides to the absolute alcohol and let it sit for several minutes

12.  Move slides to the clear safe

13.  To prepare cover slips, separate them and lay them out to be easily grabbed

14.  Apply paramount to slides with a q-tip. Remove slide from clear safe and with the brain side facing up, apply the paramount to the bottom edge facing away from you

15.  Take a cover slip and place it perpendicular to the slide until it is flat with the slide. Repeat for the remaining slides and leave slides out on a flat surface to dry