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Hi all,
I have been having problems with the carbocyanine nucleic acid stains PO-PRO-1 and YO-PRO-1, and the neuronal marker Neurotrace. We have found that when PO-PRO-1 (1:10000 in 0.01% PBST) is applied to a paraformaldehyde fixed slice of brain tissue, it localizes in the nucleolus and the cytosol similar to neurotrace. However we have found that when antigen retrieval was preformed (5 min in citrate buffer at 95 degrees) the YO-PRO-1 localizes to the nucleus only. We have also found that when neurotrace is applied at 1:200 in 0.01% PBST it localizes correctly to the cytosol of neurons, but when antigen retrieval was performed it localized to the nucleus.
Has anyone seen something like this before? Should we be concerned with over-fixing our tissues for use with YO-PRO-1/PO-PRO-1? Does the tissue require more permeabilization than just 1% triton X-100 in PBS for 2 hours at RT? Also does goat serum affect the fluorescence of neurotrace?
Any help would be great.
Thanks!
Leslie Rietveld
Department of Biology
University of Victoria, Canada
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