We have recently encountered an issue where staining for EdU on tissue sections will show EdU+. However, if this is followed by a second Ab against a protein epitope (i.e. insulin) only EdU will show up but the protein staining will not. If we switch the order Insulin 1st then EdU staining, we only get insulin expression and not EdU staining and obviously either one individually will show up just fine. Is there something in the Rxn buffer for EdU staining that would cause this issue? Any thoughts?
Dear Tyler, I am a bit confused when you say 'second antibody' because EdU staining is normally performed without antibody and the reaction is based click chemistry. I have never combined EdU staining with labeling a specific protein, but as the problem is present in both ways, this suggests problems with the treatment before putting on the Ab. Maybe the post-fixation step in the EdU staining masks the antigen for the insulin antibody? Or maybe your blocking buffer is not ok? I also don't know the Rxn buffer, so it is difficult to provide a straigth-forward answer. The combination should work though. Or you could swith back to the BrdU staining, a protocol that differs and uses an antibody-based staining technique.
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