Nissl Staining Procedure

Obtain 6 containers.

To the first container, add distilled water.

To the second container, add 70% alcohol.

To the third container, add absolute alcohol.

To the fourth container, add Clear Safe.

To the fifth container, add cresyl violet stain.

Take your slides, ensuring the back side is facing outwards, and place them in the first container.

Leave them in the first container for approx. 2 minutes before moving on to the next step.

Take the slides out of the first container and place them into the second container for 2-4 minutes (closer to 4).

Move the slides to the third container for about 5 minutes.

Move the slides into the Clear Safe. You may see smoke in the Clear Safe liquid. This means that your slides are not fully dehydrated and must be moved back into the alcohol.

Leave the slides in the Clear Safe for approx. 5 minutes, or until the slides are fully clear. 

Move the slides into the absolute alcohol for about 4 minutes, then the 70% alcohol for 4 minutes, and the water for another 2 minutes

Place the slides into the stain for about 20 minutes.

Add acid alcohol to the 6th container. Place this container between water and 70% alcohol.

Ensure your waste container is nearby. Move the stained slides into the water. As the water changes color, dump the water and refill it. 

After sitting in the water for a few minutes, move the slides into the acid alcohol, dumping it out after it changes color and refilling it quickly.

Move the slides from the acid alcohol into the 70% alcohol for a minute or so. 

Move the slides into the absolute alcohol and let them sit for several minutes.

Move the slides into the Clear Safe for a couple of minutes.

Prepare coverslips.

1. 1. Lay out the necessary number of coverslips.

   2. Open paramount and place a one-sided Q-tip in the bottle.

Grab a slide and make sure the brains are pointing away from you at an angle.

Take the paramount on the stick to carefully apply it to the bottom edge facing away from you. 

Take a coverslip and place it perpendicular to the slide about halfway down the cover slip. Move it up at a decreasing angle until it is flat with the slide. Tap on each slide to make sure it is even with the slide.

Do this for the remaining slides. You may leave the slides out on a flat surface to dry.