Staining and Stuff

Sectioning Proceedure:

1. Cool the stage until it is completely frozen by opening the valve for CO2

2. To keep the brain level and in-place, cut across the cerebellum

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

4. When the ethanol is half frozen, add a 30% sucrose layer to this base.

5. When this is frozen halfway, place the with the dorsal side facing the blade.

6. Surround the brain with sucrose solution to freeze the tissue.

7. Position the stage so the tissue is below the blade path.

8. Tighten the screws to secure the blade in its proper alignment.

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

10. Cut the tissue to user specifications.

11. When finished, remove the blade, and carefully wash with soap and water, and dry thoroughly. Coat the blade with oil to prevent rusting.

12. To mount your section, place in the dish of water, float to the top and put the slide under the tissue in the water.

13. Guide the tissue onto the slide with the brush.

14. After mounting, allow the tissue to dry before staining.

Staining Procedure:

1. Nissl Stain

   1. Obtain 6 Containers

      1. Add distilled water

      2. add 70% alcohol

      3. add absolute alcohol

      4. add clear safe

      5. add cresyl violet stain

      6. add acid E+OH

   2. Facing the back with the glass outward, place the slide into the first container. Place each slide in its place and leave for 2 minutes.

   3. Take the slides from the first container and place into the second container the same way as before. Leave for 4 minutes to dehydrate the tissue.

   4. Place the slides into the third container for 5 minutes to complete dehydration.

   5. After the 5 minutes, place the slides into the 4th container of clear safe. If what appears to be smoke in the liquid, dehydration was not complete, and container three must be repeated. If not, leave slides in container 4 for 5 minutes.

   6. Slides should be more transparent, meaning they are ready to go back to container 3 for 4 more minutes, then container 2 for 4 minutes, then container 1 for 4 minutes.

   7. After completing these steps, place the slides into the cresyl violet container. Leave the slides in the stain for 20 minutes.

   8. Taking these slides, place into container 1. Then transfer to acid E+OH. Move to container 2, then into container 3. Allow slides to sit for several minutes before transferring to clear safe.

   9. Add permount to the slides with the brains facing upwards to prepare for cover slips. Add this to the bottom edge of the slide that is facing away from you.