Sectioning and Staining

Nissl staining: 

• Overview

  • Stains cell bodies (nissl bodies)

  • takes ~1 hour per 5 slides

  • takes 13 steps 

• steps: 

  • Setup

    • Obtain 6 containers and line them up in a row with the 6th container at the back 

    • 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 

  • 1. Place slides into the first container with distilled water (make sure the back slide with the glass is facing outwards); place each into the designated slot in the container and leave for ~2 minutes 

  • 2. Take slides out of the distilled water and place into container with 70% alcohol for ~2-4 minutes to dehydrate the tissue 

  • 3. Move slides into absolute alcohol to finish the dehydration process, leave in for ~5 minutes 

  • 4. Move slides into the clear safe; you may see something like smoke in the liquid when placing the slides in. If this occurs, the tissue hasn't been properly dehydrated and needs longer in the absolute alcohol before moving on. If no smoke occurs, leave tissue in for ~5 minutes or until tissues are fully cleared 

  • 5. Take slides out of clear safe, and if slides are more transparent then they were before, they can go into the absolute alcohol for ~4 minutes 

  • 6. Move slides into 70% alcohol for ~4 minutes

  • 7. Move slides into the distilled water for ~2 minutes 

  • 8. Place slides into cresyl violet stain for ~20 minutes 

  • *Prep work* - add 10% acid alcohol to the 6th container that had been in the back 

    • Place acid container in line with the other five containers, between the water and the 70% alcohol -- it will be needed in process of getting from the stain back to the clear safe 

    • Have waste container nearby 

  • 9. Move slides from stain into water (the water will change color, dump out the water and refill it); let sit in water 

  • 10. Move slides into acid alcohol and repeat process- once the acid alcohol changes color, dump it out and refill quickly to not dehydrate tissue

  • 11. Move from acid alcohol into 70% alcohol; leave for as long as you see necessary 

  • 12. Move into the absolute alcohol; let sit for a bit longer 

  • 13. Move into clear safe; it should stay here until visible that it isn't losing stain anymore

  • 14. Prepare cover slips; use clean glove or no gloves- separate cover slips (if you see a rainbow sheen, that means multiple are stuck together)

    • gather as many cover slips as you need for slides

    • carefully lay them out so that you can easily grab them 

    • put gloves back on to prepare to cover slip; open permount 

    • use one sided q-tip placed inside the permount to apply permount to slides 

    • remove slide from clear safe; have side with brains facing upwards 

    • hold at angle facing away from you 

    • apply permount to bottom edge that faces away from you 

    • place cover slip perpendicular to the slide about halfway down the cover slip; move upwards at a decreasing angle until it is flat with the slide 

    • tap on each side to assure that it is even with the edges of the slide 

    • repeat for each slide 

    • leave slides out on a flat surface to dry 

Tissue sectioning: 

• steps: 

  • First open valve to CO2 tank, and cool stage until completely frozen 

  • Cut straight across cerebellum so brain can stay level and upright 

  • Apply 1% Ethanol to frozen stage as the base 

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

  • When Sucrose is halfway frozen, place brain so that the dorsal side will face towards the blade 

  • Add more sucrose around the tissue and allow to freeze 

  • Manually lower or raise the stage so that the tissue is below where the blade will run 

  • Place 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 distilled H2O to place tissue 

  • Begin cutting tissue into sections of the desired thickness (50 microns) and fully extend cutting arm for automatic adjustments 

  • When done cutting, remove blade, wash with soap and water, then dry. Coat blade in oil and store until next use. 

  • For mounting, place one section in dish of water, 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