Hello,
I have an issue with my Alizarin Red staining. It gives random unspecific positivity stains throughout the tissue (Embedded in paraffin, dewaxed in Xylene). My question is, how do I remove these unspecific stains, or how can I remove the staining altogether?
Thank you.
With other stains I would try to wash the slide with acidified ethanol, but acid would probably solve the calcium, that you want to detect.
The pH of the staining solution has to be at pH 4,2 for a specific calcium-staining. Check the pH and adjust with 0,5 % ammonium hydroxid if necessary. (reference: Utah staining protocol, Freida Carson histotechnology)
Alizarin Red S Solution:
Alizarin red S 2.0 gm
Distilled water 100.0 ml
Mix the solution, adjust the pH to
4.1-4.3 using 0.5% ammonium
hydroxide. The pH is critical, make
fresh.
Maybe you can try to destain as is typically done using 10% CPC for 15 min with rotation @ RT. You can usually quantify these by transferring 200ul of the diluted sample into 96 wells and reading at 562.
10% CPC = 1 g Cetylpyridinium chloride CPC in 10 ml of Sodium Phosphate 10mM pH 7
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