I want to check aorta for changes in protein expression in endothelial cells using microscopy. My primary antibody is FITC tagged and elastin is giving strong green fluorescence which makes it hard to get clear picture of endothelial cells.
Generally speaking your best bet is to get another label on your Ab, or try a different label on a secondary antibody.
Alternately, there are some procedures using blue dyes (e.g. Pontamine sky blue) as counterstains to shift the autofluorescence to the red part of the spectrum.
I recommend this primer from the Wright Cell Imaging Facility, Toronto Western Research Institute:
http://www.uhnresearch.ca/facilities/wcif/PDF/Autofluorescence.pdf
They have a plethora of information on their site:
http://www.uhnresearch.ca/facilities/wcif/
Generally speaking your best bet is to get another label on your Ab, or try a different label on a secondary antibody.
Alternately, there are some procedures using blue dyes (e.g. Pontamine sky blue) as counterstains to shift the autofluorescence to the red part of the spectrum.
I recommend this primer from the Wright Cell Imaging Facility, Toronto Western Research Institute:
http://www.uhnresearch.ca/facilities/wcif/PDF/Autofluorescence.pdf
They have a plethora of information on their site:
http://www.uhnresearch.ca/facilities/wcif/
Thank you Peter for the valuable information. I agree going with new tag should help.
We had similar problem while staining lung tissue and had to avoid FITC. Peter, thanks a lot for the links, they are very useful!
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