Carmine Stain Protocol (adapted from rat procedure by Amy Moser by Lisa Arendt)

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Lisa’s Carmine Stain Protocol

Carmine stain preparation

  • 2.5 g Alum potassium sulfate
  • 1.0 g Carmine (Sigma C-6152)
  • to 500 mls dH20
  • Boil for at least 40 minutes and keep hot. Filter through Whatman #1 paper and adjust to final volume of 500 mls. There will be a lot of stain that doesn’t dissolve.  This can be minimized by keeping the solution boiling while filtering a small amount at a time.

Fixing and staining

  1. Spread glands on a glass slide and allow to sit for ~10 min or so until they become ‘stuck’ to the slide.  All fixing and staining procedures can then be done in a coplin jar. Alternatively, flatten glands between glass slides during formalin fixation (do not allow to stick to glass) and then do all staining procedures with glands in tissue cassettes.
  2. Fix glands in 1:3 glacial acetic acid: 100% EtOH for 1 hour or formalin fixation overnight is fine.
  3. Transfer to 70% EtOH for 15 minutes followed by a short rinse in 50% EtOH (5 min) then dH20. (If fixing in formalin, a wash in dH20 should suffice). Following formalin fixing, glands can stay in 70% EtOH until you are ready to stain them.
  4. Stain in carmine for 1-3 days.  Fatter glands may take more than 3 days.
  5. Dehydrate in 70% EtOH overnight, then 90% and 100% EtOH with 60 minute washes.
  6. Transfer to xylenes overnight to clear the fat from the gland. Fatter glands may take a few days to clear.
  7. Wash with 100% EtOH to remove the xylenes.
  8. Transfer to glycerol (or mineral oil or methylsalicylate) in a clean coplin jar. Store the glands in glycerol in open coplin jars in the hood for a few days until the xylenes are truly gone (a couple of days).
  9. If the carmine is too dark, the glands can be rehydrated through graded alcohols to 70% EtOH and remain until some of the stain precipitates out of the glands.
  10. Visualize glands with coverslips over the tissue, use additional glycerol to fill the spaces around the glands (note, this will be a bit messy…make sure to clean up the microscope after use!).  If wanted, glands can be mounted with permount for long term storage.
  11. To submit for sectioning, scrape gently off of the slides and transfer to tissue cassettes for processing.