Sectioning & Staining 

Sectioning 

• Open the valve to the tank of CO2 and cool the stage until it is completely frozen. 

• To make sure that the brain stays upright and level, cut across the cerebellum. 

• As the base, apply 1% ethanol to the frozen stage. 

• When the EtOH is halfway frozen, add a layer of 30% sucrose to the base. 

• When the sucrose is frozen halfway, place the brain where the dorsal side faces the blade. 

• To allow the tissue to freeze, add sucrose around it. 

• Lower or raise the stage so that the tissue is below where the blade runs. 

• Place the blade in its designated space, and tighten the screws to hold the blade in place. 

• Obtain a brush to handle the tissue, and a dish full of H2O to place the tissue. 

• Cut the tissue into sections of the desired thickness. Lower the blade as needed. 

• When finished cutting, remove the blade, wash with soap and water, then dry. Coat the blade in oil and store it for next use. 

• For mounting, place one section in a dish of water, float to the top, and place the side under the tissue in water. 

• Using a brush, guide the tissue along the side. 

• When finished mounting, allow the tissue to dry before staining. 

Staining 

• Nissl stains stain for cell bodies 

• 6 containers 

  • To container 1 add distilled water 

  • To container 2 add 70% alcohol 

  • To container 3 add absolute alcohol

  • To container 4 add clear safe

  • To container 5 add the cresyl violet stain

  • To container 6 add acid EtOH

• Making sure that the backside with the glass is facing outward, place the slide into the first container. Place each slide into its designated slot and leave them for approx. 2 min. 

• Next step, take the slides out of the first container and place them into the second container. Leave them for 4 min. to properly dehydrate the tissue. 

• After, take the slides and move them into the third container to complete the dehydration process. Leave them in there for 5 min.

• Move the slides into the fourth container. When the slides are placed into the clear safe (fourth container), if what looks like  "smoke" appears inside of the liquid, that means the samples are not fully dehydrated and you must place them back into container 3. Otherwise, keep the slides in the clear safe for 5 min. 

• Steps 5-8 are the reversal of the process that was just done. When the slides are taken out of the clear safe, they will be slightly more transparent which means it is ready to go back into container 3 for approx. 4 min, then into container 2 for another 4 min, and then container 4 for 4 min. 

• Finally, you can place the slides into the cresyl violet stain. Leave the slides in the stain for about 20 min. 

• After the 20 min is up, place the slides into container 1. Move the slides into the acid EtOH. Then move the slides into container 2 and then into container 3 (leave them in there for several min before moving them to the clear safe). 

• When preparing the cover slips, add permount into the slides (brains facing upwards). Add the permount to the bottom edge of the slide that is facing away from you.