Staining Procedures

Sectioning

1.Open CO2 tank valve, cool stage until fully frozen

2. Make a straight cut across cerebellum ensuring brain stays upright/level

3. Use 1% ethanol as the base for the frozen stage

4. When ethanol is partially frozen, add 30% sucrose as the base

5. When sucrose is partially frozen, place the brain facing the dorsal side to the blade

6. Add additional sucrose and allow to freeze

7. Adjust the stage by lowering or raising it so tissue is below where the blade will run

8. Place blade in correct position, tightening screws to hold in place

9. Obtain brush and a sectioned dish of distilled water to place the tissue

10. Cut tissue into sections ~50 microns~ extend cutting arm for automatic adjustment

11. After cutting is finished, remove and wash the blade with soapy water. Dry and store it in oil.

12. Place a section in a dish of water, allow to float to top, place slide under the tissue in the water when mounting

13. Guide tissue to correct spot on slide using a brush

14. Allow tissue to dry after mounting 

Staining

1. Obtain 6 containers

2. Distilled water->> container 1, 70% alcohol->> container 2, absolute alcohol->> container 3, Clear Safe->> container 4, cresyl violet stain->> container 5

3. With the backside facing outwards, place them in container 1 for 2 minutes

4. Place the slides into container 2 for 2-4 minutes

5. Place slides into container 3 for 5 minutes

6. Place the slides into container 4. A smoke may be present which means the slides should be placed back into alcohol for additional dehydration

7. Let the slides process for 5 minutes or till fully clear

8. Place slides back into container 3 for 4 minutes. Then put them into container 2 for 4 minutes. Then container 1 for 2 minutes.

9. Put slides into the stain for 20 minutes

10. Add acid alcohol to container 6. Place container between water and 70% alcohol.

11. With the waste nearby, move stained slides into the water. When water changes color, dump into waste and refill with clean water. 

12. After a few minutes, place slides into acid alcohol. Dump the alcohol as it changes color and quickluy refill.

13. Move slides into 70% alcohol for about a minute.

14. Let slides sit in absolute alcohol for several minutes.

15. For 2-3 minutes, let sit in the Clear Safe.

16. Prep the coverslips: Lay out proper number of slips and place q-tip into the paramount bottle

17. Grab slide, ensure brain is pointing away from you at an angle

18. Using the paramount q-tip , apply it to bottom edge, facing away from you.

19. Place coverslip perpendicular to slide (halfway down the cover slide). Make sure it is flat with the slide. Tap to ensure eveness.

20. Leave the assembled slides on a flat drying surface.