Immunfluorescence analysis of rat artery

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Immunfluorescence analysis of rat artery

Postby fluro » Aug 08 2015 2:37 am

I am trying to do some co-localization studies in rat cerebral artery using cryosectioning. For the same, I am optimizing individual antibody at first.

My protocol is as follow: Incubate the sections with blocking solution (3-10% BSA) 1 hr, Primary Antibody (in 1.5-5% BSA) overnight, fluorescence conjugated secondary antibody (in 1.5-5% BSA) for 1 hr with TBS washing in between. My protocol does work & I do see fluorescence in specific filter.
But, microscopy specialist always suggest the fluorescence is not specific by doing following test:
Suppose I have FITC conjugated section & I see the fluorescence in FITC filter. He scan the same tissue section in Far-red (Cy5/ Texas red) filter & images the tissue there. If he see some emission there, he suggest that it is not specific. He also overlaps the images from FITC & Cy5 and suggest it is non-specific.

I am not sure it is correct way to do it? Because I spoke with few people & they said to take images in specific filter (like FITC) without considering other filter as some artifacts would be always there.
I tried several protocols but the problem always persists. Does anyone has similar experience? Can you please suggest possible remedies
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