/wp-content/uploads/2015/09/kuperwasser-banner-500px-wide-no-bg-semi-bold.png 0 0 admin /wp-content/uploads/2015/09/kuperwasser-banner-500px-wide-no-bg-semi-bold.png admin2015-09-03 14:08:362015-09-03 14:21:29Carmine Stain Protocol (adapted from rat procedure by Amy Moser by Lisa Arendt)
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
- 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.
- Fix glands in 1:3 glacial acetic acid: 100% EtOH for 1 hour or formalin fixation overnight is fine.
- 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.
- Stain in carmine for 1-3 days. Fatter glands may take more than 3 days.
- Dehydrate in 70% EtOH overnight, then 90% and 100% EtOH with 60 minute washes.
- Transfer to xylenes overnight to clear the fat from the gland. Fatter glands may take a few days to clear.
- Wash with 100% EtOH to remove the xylenes.
- 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).
- 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.
- 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.
- To submit for sectioning, scrape gently off of the slides and transfer to tissue cassettes for processing.