SnS

Sectioning and Staining Assignment 12/7/23

Microtome Step-by-Step:

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

2. Make a cut straight across cerebellum so brain can staylevel and upright

3. Apply 1% ethanol to 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 brain so that the dorsal side will face towards the blade

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

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

8. Place 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 H2O to place tissue

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

11. When done cutting, remove blade, wash with soap and water, the dry. Coat blade in oil and store until 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 side

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

15. Done

Nissl Stain:

1. 6 containers, (1st: Water, 2nd: 70% Alcohol, 3rd: absolute Alcohol, 4th: Clear safe, 5th container: Cresyl Violet Stain)

2. Take slides and put it into each container for 2 minutes

3. Take slides out, and place them into the 70% alcohol for 2-4 minutes

4. After 4 minutes, remove from 70% alcohol and more to the absolute alcohol, leave it in for 5 mintutes

5. Take it out of the absolute alcohol, put it into the clear safe

6. Repeat steps 2-5 in reverse order

7. Place into the violet stain for about 20 minutes

8. While slides are in stain, prepare last container with Acid EtOH, then place it between water and 705 alcohol.

9. Move slide into water after 20 minutes. After water changes color, dump it and refil it.

10. Then place it in the EtOH Acid Alcohol and after changes color, dump and refil.

11. Repeat those steps in the 70% alcohol and clear safe. At this point, the color of the liquid shouldn't change.

12. Grab 3 cover slips, open sticky paramount and apply it to the slides while facing away from you.

13. Do this with the rest of the slides.