Talk:Kubke Lab:Protocols/Nissl Stain Protocol

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Reagents:

0.1% Cresyl violet solution:
Chemical Amount
Cresyl echt violet (or cresyl violet acetate) 0.1 g
Distilled water 100 ml

Method

NB: Staining using cresyl violet solution heated to body temperature may increase stain penetration.

  1. Place the section-mounted slides into a slide basket/tray so they are standing vertically.
  2. Place the tray with the slides into a container filled with 70% alcohol such that the sections are completely covered for 5 minutes.
  3. Next place the tray into a 95% alcohol solution for 5 minutes followed by a 100% alcohol solution in the same manner for 5 minutes.
    NB: Ensure that the tray is free from solution and as dry as possible without allowing the sections to dry before placing them in the next dehydration battery container. This will prevent the alcohol solutions from losing their purity.
  4. Then place the tray into xylene for 15 minutes.
  5. Rehydrate the slides following soaking in xylene to allow for stain penetration. Instead of 5 minutes keep the tray in the alcohol containers for 2-3 minutes each starting from 100% > 95% > 75% and finally dip the tray into a container of tap water for 5 seconds.
  6. Soak the tray and slides in a solution of Crystal Violet until the desired degree of staining is obtained, this will be between 5-15 minutes.
    NB: If the staining is too dark then the slides will need a longer time in the alcohol solutions again during dehydration however if the staining is too light it will need more time in the stain before dehydrating again.
  7. Once the premium staining level is reached the tray needs to be placed through the dehydration battery for a final time, this time leaving the tray in the containers for about 1 minute each (Water > 70% alcohol > 95% > 100% and finally soaking in xylene.

At this point the slides are ready for a coverslip to complete the staining procedure.

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