I did an immuno ISH using a DIG-labeled antisense RNA probe and my antibody of interest. The DIG signal was obvious when it came time to wash the slides and add primary antibody to them. I know that sometimes the signal weakens, but it should never disappear. After mounting the slides using CFM-1 Plus mounting medium, the immuno stain was visible under fluorescent light, but the DIG reaction as no longer visible under white light. I was not even able to find the sections on my slide. Normally, I use Slow-Fade Gold and never had this problem. Thank you!
Hi Richard,
I suspect it is the Glycerol in the mounting medium, which causes the staining to fade. NBT/BCIP stain or Fast Red tend to "bleed" when subjected to Glycerol. This can be prevented by postfixing the sections once the in situ hybridization is complete. But I am not sure, what effect that would have on the IHC, though. Maybe just use a mounting medium, which does not contain Glycerol.
Best Simone
Hi Simone, I don't think that it is the glycerol. I use regularly Glycerol mounting medium to mount ISH DIG slides and the signal is fine, so it must be something in the CFM, other than glycerol, that is causing the DIG signal to disappear. It is not just faded. The signal is completely gone, including background.
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