One antibody stains soma and the other stains a diffuse protein, located throughout the cells in soma and neurites. We don't see fluorescent overlap, but how can I quantify this? The protein antibody does not express as clearly outlined cells, thus it does not seem feasible to do cell counts.
There is a number to measure coloc that goes from -1 to 1. 1 is 100% coloc, 0 is no coloc and -1 is negative coloc (100% segregation). You can read more in: A guided tour into subcellular colocalization analysis in light
microscopyS. BOLTE* & F. P. CORDELIÈRES
Journal of Microscopy, Vol. 224, Pt 3 December 2006, pp. 213–232
Received 13 April 2006; accepted 28 June 2006
Can you stain cell membrane to have a clearer outline of the cells so that you can quantify the population co-expressing your targets?
Can try quantitative analysis - Intensity. The fluorescence analysis software should have this feature. Mine is NIKON NIS Element. Measure the expression intensity. You can select the region of your interest - the diffused regions, save it and reapply to your subsequent images. Sounds complicated. We have a dedicated NIKON Imging expert to help us with this.
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