I've posted a question regarding my IHC results previously, whereby my tissue of interest was having autofluorescence and gotten some revisions to my IHC protocol. However, I realized that the tissue without having primary nor secondary antibodies added to it already had autofluorescence. That tissue I'm studying also contains collagen and elastin, which contributes to autofluorescence, and was fixed for more than a month in formalin. I've read that the addition of sodium borohydride can reduce formaldehyde-induced autofluorescence.
Besides over fixation, could the decalcifying reagent have a role in autofluorescence? I used 30% formic acid, and was advised to try Morse's solution, which is 20% formic acid with 10% sodium citrate.
I'll try performing IHC on samples I've harvested and fixed for 72hrs and 24hrs, in either formalin or formaldehyde. Has anyone used either 30% formic acid or Morse's solution as the decalcifying reagent and which is better?
I was not successful using fluorescent antibodies on decalcified tissues due to autofluorescence.
I have used 10-14% EDTA for decalcification or immunodcal solution (see reprint) which is a mild formic acid, I was able to get immunohistochemistry of collagen I, Collagen II, Aggrecan, Lubricin, Collagen VI etc. Only trick is to to optimize antigen retrieval method for each antigen (see the reprint)
Sodium borohydride may help to reduce the background noise. But at the same time it will mask your target signal and make it weaker too.
I will suggest to use heat based antigen retrieval with 10mM sodium citrate buffer (ph 6.0). And use EDTA based decalcification suggested by Dmitry Zinovkin.
When it comes to using decalcifying agents, see what is commonly employed in literature for your tissue of interest. The more calcium, the longer it takes to decalcify. More acidic decalcifying agent, the greater the chance of epitopes denaturing. As I've mentioned in another post, I've long completed my Masters and have also found a sweet spot being using 15% EDTA solution on the rat lumbar spine over a month with agitation and occasionally switching out for a new solution.
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