Sectioning and Staining Procedure 

Tissue sectioning procedure:

1. Open valve to the CO2 tank, and cool the stage until totally frozen 

2. Cut straight across the cerebellum so the brain can stay level and upright

3. Apply 1% ethanol to the frozen stage as the base 

4. When EtOH is halfway frozen, add a layer of 30% sucrose to the base 

5. When Sucrose is halfway frozen, place the brain so the dorsal side faces toward the brain

6. Add more sucrose around the tissue and allow to freeze

7. Manually lower or raise stage so the tissue is below where the blade will run 

8. Place the blade in its designated space and tighten screws to hold the blade in place

9. Obtain a brush to handle the tissue and sectioned dish full of distilled H20 to place tissue 

10. Begin cutting tissue into sections of the desired thickness (50 microns) and fully extended arm for automatic adjustments 

11. When done cutting, remove blade, wash with soap and water, then dry. Coat blade in oil and store for next use.

12. For mounting, place one section in dish of water, float to the top and place slide under tissue in water

13. Using a brush, guide the tissue to its spot on the slide

14. When done mounting, allow tissue to dry before staining 

Nissl staining procedure: 

1. Obtain six containers with the sixth placed in the back 

2. Add distilled water to container #1

3. Add 70% ethanol to container #2

4. Add absolute alcohol to container #3

5. Add Clear Safe  to container #4

6. Container #5 (placed in the front) will contain cresyl violet

7. Place each of the slides into the distilled water for two minutes 

8. Place the slides in the 70% ethanol to dehydrate the tissue for four minutes

9. Place the slides in the absolute alcohol for 5 minutes

10. 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 

11. Again, place the slides in the absolute alcohol for 5 minutes

12. Again, place the slides in the 70% ethanol to dehydrate the tissue for four minutes

13. Again, Place each of the slides into the distilled water for two minutes 

14. Place the slides into the cresyl violet stain Leave the slides in the stain for 20 minutes 

15. Prepare container #6 with acid alcohol 

16. Place container #6 in between the water and the 70% alcohol

17. Move the slides out of the stain and into the water (refill water)

18. Then move into the acid alcohol. Once there is a color change refill the acid alcohol 

19. Then move the slides into the 70% alcohol (eye ball the color

20. Move the slides into the absolute alcohol container for several minutes

21. Then move the slides into the Clear safe container (you shouldn’t lose stain after this step) 

22. Prepare the cover slips (one for each slide)

23. Apply paramount onto the slides with a q-tip 

24.  Apply paramount to the side of the slide facing away from you 

25. Place coverslip perpendicular to the slide 

26. Tap on each side to ensure they are properly aligned 

27. Repeat for the remaining slides