I am staining for CD163 in human tissue embedded in paraffin. I expect to find most of my staining in the perivascular space, but I cannot get rid of blood autofluorescence.
Quenching autofluorescence is really tricky especially in nervous tissue and on erythrocytes. I have had some success using the Sudan Black B protocol as shown in this paper "Control of autofluorescence of archival formaldehyde-fixed, paraffin-embedded tissue in confocal laser scanning microscopy (CLSM)."Baschong W1, Suetterlin R, Laeng RH.
Another trick is to use a fluorophore that has an emission spectrum at a longer wavelength, for example 649NM or higher. Longer wavelength usually corresponds to lower autofluorescence. Hope this helps!
Thank you, Michael,
I did Sudan Black at 1% for 5 min, and it reduced the background, but not the vessels. I will try adding the ammonia step from the paper you referenced.
can you take an autofluorescence image before staining and subtract that with software from the image after staining? This is how it is usually done. It only requires that you should reposition your sample exactly.
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