Technique / Histology / Immunohistochemistry techniques

Immunofluorescent staining autofluoresence problem

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By Rob on 02-Mar-2008 Does anyone out there have suggestions for reducing/eliminating autofluorescence in paraffin-embedded kidney tissue?!? I've tried both formalin and Histochoice for fixation (both glow brightly, the formalin a little less), I've treated with sodium borohydride (no effect), and I recently tried blocking with egg white and skim milk (to block biotin - no effect 1st time), but I'm going to try that again before giving up. The autofluorescence is primarily inside the cells, so I figure it's biotin, and not really a collagen or elastin problem. Since the Histochoice samples were just as bad as the formalin, I'm guessing it's not primarily an aldehyde thing (supported by the lack of results from the borohydride). It fluoresces with our DAPI, FITC, and TRITC sets, and I see it with and without BSA blocking. I've given up on trying to stain with antibodies until I can diminish or eliminate the autofluorescence. Is there any hope?!? Rating: n/a Reply