I want to asses vascular calcification in a mouse model of Chronic Kidney Disease. I am planning to have part of the aorta fixed in 4% PFA overnight and then embedded In paraffin (FFPE) and later stain the sections with Alizarin Red. Another part of the artery fixed in PFA overnight and stained with Alizarin Red (whole mount).
I have tried to look for protocols for this staining but I have found contradictory information on what could be the best protocol.
For the FFPE sections, I have found that the pH should be around 4,1 and 4,3 but some use KOH, some use NH4OH, some others use NaOH to adjust the pH. Does it make any difference?
At what concentration should I use Alizarin Red S (I have found between 0,5 to 2%)?
For the whole mount, I have found that I should place the tissue in 0,003% Alizarin red for 30 hours and some that use 0,5% Alizarin Red for just 20 minutes. Can you recommend a protocol?
I have no practical experience with Alicarin red, but for me it doesn't matter which base you use as long as there is no calcium added.
For the correct concentration I would pick one in the middle and stain a serie of known positive tissue. Additional compare with vonKossa-stain. This is a reliable caliumcarbonat-proof. The ready-made solution from Merck is about 1,3 % and has a pH about 4,1.
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