Steps for Tissue Sectioning:
-open valve to CO2 tank
-allow the stage to cool until frozen
-cut straight across the cerebellum to keep the brain level and upright
-apply 1% ethanol to frozen stage as the base
-after EtOH has frozen halfway, add a layer of 30% sucrose to the base
-when the sucrose is halfway frozen, place the brain so that the dorsal side will face towards the blade
-add more sucrose and allow the tissue to freeze
-manually lower or raise the stage so that the tissue is below where the blade will run
-place the blade in its designated space and tighten screws to hold the blade in place
-obtain a brush to handle the tissue and sectioned dish full of H2O to place tissue
-begin cutting tissue into sections of the desired thickness and full extend cutting arm for automatic adjustments
-once finished cutting, remove blade, wash with soap/water, dry. Coat blade in oil and store until next use
-for mounting, place one section in water dish, float to the top and place slide under tissue in water
-using a brush, guide the tissue to its spot on the slide
-when done mounting allow your tissue to dry before staining
Steps for Nissl Staining
-Obtain 6 containers
-to the first, add water, the second 70% alcohol, 3rd absolute alcohol, 4th clearsafe, and 5th cresyl violet stain
-STEP 1: Make sure the backside of the slides is facing outwards, place each of your slides into their designated containers(distilled water)
-STEP 2: Remove slides and place them in the container with the 70% alcohol(2-4 minutes)
-STEP 3: Remove the slides, and place them in the container with the absolute alcohol(5 minutes)
-STEP 4: Remove the slides and place them in the container containing the clearsafe. If smoke appears, remove the tissue, and dehydrate it in the alcohol again.
-STEP 5: Remove the slides, and place them in the absolute alcohol(4 minutes).
-STEP 6: Remove the slides and place them in the 70% alcohol(4minutes).
-STEP 7: Place the slides in the distilled water for 2 minutes.
-STEP 8: Place your slides in the cresyl violet stains(20 minutes).
-INTERMEDIATE STEP: Fill a new container(sixth) with acid alcohol
-STEP 9: Move the slides to the water. Dump out the water and refill it.
-STEP 10: Place the slides in the acid alcohol. Dump it out and replace it.
-STEP 11: Place the slides into the 70% alcohol.
-STEP 12: Place slides in the absolute alcohol. Allow it to sit for a bit.
-STEP 13: Place the slides in the clearsafe.
-After all of these steps, prepare the coverslips with paramount, and apply the slides to them. Allow the cover slips to dry now and the process has been completed.