In the lab we fix joints for 24 hr in 4% PFA, followed by decalcification in 50% formic acid/PFA for 2-3 days prior to paraffin-embedding.
For tissue sections other than joints, we perform antigen retrieval using heat-mediated antigen retrieval (boiling in citrate buffer for 3-9 minutes, depending on the tissue type). When applying this retrieval method on joint sections, the sections detach from the slides. We also tried PIER using 0.1% trypsin, 10-20 minutes at 37°C, but no luck.
We are interested in antigen retrieval for collagens, MMPs and infiltrating T- and B- cells in joint tissue of arthritic mice.
Does anyone have experience with antigen retrieval of any of these antigens in PFA-fixed paraffin-embedded mouse joint sections? I would be really grateful if somebody could share their working protocol.
I haven't experience for these antigens or tissue, but my sections don't detach from the slides when I started to use the commercial citrate buffer (pH 7,4 or 6,0) instead of the "home made" ones.
Good luck!
Epitope retrieval post decalcification may be difficult for certain epitopes. The success of antigen retrieval protocols in tissues fixed with buffered formalise and embedded in paraffin and acid decalcified is dependent upon the antigen (epitope) you are attempting to identify. For example carbohydrate antigens such as Lewis x, CEA, CA 19.9 are acid stable and can be more successfully exposed using low pH methods and proteases whereas high pH techniques are of little or no value with these epitopes. In contrast, protein epitopes may be successfully exposed by incubation with chelators (citrate & EDTA) in buffers pH 6 - 8. However from your comments above chelation resulted in your section floating from the slide. You may need an anchor layer such as poly L lysine. I have used slides pre coated with poly L lysine, fixed with excess glutaraldehyde (1%) wash and dried to anchor foetal joint sections to slides. Also please note that acid treatment of collagen hydrolyses it to gelatine. Your epitopes on collagen may be destroyed. You may wish to try a different decalcification method which will not affect gelatine. You may wish to consider chelation methods. See: http://www.leicabiosystems.com/pathologyleaders/an-introduction-to-decalcification/ I have attache a document that may provided options you may choose to employ should your results not be satisfactory.
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