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Alizar Red for calcium - (Jan/11/2010 )


I have to stain cryosections with Alizar Red to detect calcium. I would need a good protocol for it?
I've found a few so far, but not for cryosections yet.

Another question related to Alizar Red; the solution should be pH 4.1-4.3 and balanced with ammonium hydroxide. Is this critical and can I Balance with NaOH? How long the solution is preserved in +4C?



I've tried this straightforward protocol for my frozen sections:

1. Air drying 15-30min (depending section thickness)
2. Ice-cold acetone fixation 10min
3. PBS wash 5min
4. Alizarin red 1-4min
5. Wash under running cool tab water 5min
6. Mount with Aquamount

However the red stain moves from the section under the coverglass in mounting medium. So I can't get reliable result. Any suggestions to improve my protocol?