Stain and Sectioning 

Nissl Staining Procedure:

1.) Gather six different containers. In the first container you add water, to the second container you add 70% alcohol, to the third container you add absolute alcohol, to the fourth container you add clear safe, and to the fifth contain you add crystal violet stain. The sixth container will remain empty for later use.

2.) Place each slide with its glass back facing outwards into the first container. Leave the slides in there for approximately 2 minutes.

3.) After 2 minutes, place the slides in the second container containing the 70% alcohol. Leave them in there for 2-4 minutes.

4.) After, place the slides into the third container containing the aboslute alcohol. Leave the slides in there for 5 minutes. 

5.) After 5 minutes, place the slides into the fourth containing containing the clear safe. Leave the slides in there for 5 minutes. If smoke appears in the liquid, the tissue was not dehydrated enough which means that they have to be placed into the absolute alcohol for a couple of more minutes.

6.) After 5 minutes, place each slide into the absolute alcohol and 70% alcohol for about 4 minutes and in the water for about 2 minutes.

7.) Place the slides into the fifth container containing the crystal violet stain. Leave the slides in the stain for about 20 minutes. 

8.) Add acid alcohol to the sixth container while the slides are in the stain.

9.) After 20 minutes, add each slide into the first container containing the water. When the water changes color, dump the water and refill the container with clean water. Let the slides sit in the water for a couple of seconds. 

10.) Place the slides into the acid alcohol. Once you see a color change, dump out the acid alcohol and refill it with clean acid alcohol. Leave the slides in there for a couple of second. Repeat this process for the third container containing the 70% alcohol.

11.) Place the slides into the absolute alcohol and let them sit there for a couple of minutes.

12.) Place the slides into the clear safe and let them sit for a couple of seconds. 

13.) Remove the slides from the clear safe and apply permount to the edges of the slide and place a cover slip on it. 

Tissue Sectioning Procedure:

1.) Open valve to CO2 tank and let cool the stage until it is completely frozen.

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

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

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

5.) When the sucrose is  halfway frozen, place the brain on the stage in which the dorsal side is facing towards the blade. 

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

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

8.) Place the blade in place and tighten the screws to hold the brain in place.

9.) Obtain a brush to handle the tissue and sectioned dish full of distilled H2O to place the tissue. 

10.) Begin cutting the tissue into your desired thickness and fully extend the cutting arm for automatic adjustments. 

11.) Once you are done cutting, remove the blade, wash it with soap and water, and dry it. Coat the blade in oil and store it. 

12.) For mounting, place one section in a dish of water, allow it to float to the top, and place the slide under the tissue in water. Use a brush to guide the tissue on the slide. 

13.) Allow your tissue to dry.