Methods and Techniques Discussion > Immunofluorescence (IF)

Immunofluorescence, multiple staining (membrane and nuclear)


Hello all,
Can anyone offer me any insight into multiple immunofluorescent staining on blood smears for 2 membrane and 1 nuclear markers?  Should antibodies be added simultaneously?  Preferred fixation? Which permeabilizing agents are recommended (is Triton going to be too harsh for the membrane proteins I'm trying to target?).  Blocking reagents?
Thank you so much in advance!
Jennifer Campbell, HT(ASCP)cm

so what are they used for? you can tell us more details

I used triton x for a membranous stain once and I ended up with very high background and no specific stain on all processed slides in IFC. Even though one marker is nuclear, I didn't have a problem just not using the triton x when double labeling with one nuclear and one membranous. It'll depend on what your protein target is on the membrane though. Try a few slides with and without adding it and just see what happens and which result gives you the best image.


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