Fluorescence lump formation in an protein sandwich assay

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Fluorescence lump formation in an protein sandwich assay

Postby aman_1183 » May 17 2013 6:29 am

Hi All,

I am new to fluorescence microscopy. I am trying to detect a sandwich assay using FITC labeelled streptavidin. What I did was, I first put the anchoring antibody followed by the antigen and then another antibody which was biotinylated. Over this biotinylated antibody, I put FITC labeelled streptavidin. I area of intrest get illuminated as expected but along with the illuminated surface I also see alot of fluroscence lump
f sample_positive area.tif
. Could some one please tell me what could be the cause for it and how to avoid it.

Thanks
Saurabh
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