Please bear with me trying to puzzle out the following problem, it concerns in situ hybridization on paraffin embedded slides:
I am looking for expression of gene A with a probe which I know has worked for gene A previously (in whole embryos).
I know that gene A is highly expressed in the liver, and not very highly expressed in my tissue of interest.
My positive control for my ISH experiments is thus liver with anti sense (AS) probe - works perfeclty and I get a nice strong signal after 2hrs of exposure (I use NBT/BCIP with DIG labelled probe). My negative control, liver with sense (S) probe, is also clean.
However, in the tissue of interest (where expression of gene A is so low that I need to develop overnight), I see an increase of both AS probe signal, and S probe signal across various ages of my mice (not the same strength as AS, but still, the same increase over various ages).
I am really at my wits' end, since I think this can't be probe contamination, as my liver is negative with the S probe? Or can the sense probe be contaminated with AS, and not come up in the liver but in the tissue of interest? I have been doing this again, from scratch, to see if it is still the same, and how repeatable it is, but how can it be a contamination of the S probe with AS if my liver S is so CLEAN?
Please, any ideas/help highly appreciated!
Ps. Is there any (tiny) possibility that in my tissue of interest an AS transcript exists of gene A, and I am picking this up with my S probe? Or is this utterly utopic?
Edited by Maya1988, 10 July 2012 - 03:13 AM.