[Histonet] RNA in situ hybridization-staining problems

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Nicole,
 
I encountered the same problem and switched to the HRP-conjugated anti-DIG antibody also by Roche.  I used a TSA amplification system (Biogenex - mainly used for their machines, but I was able to use it by hand also) and finally, detected with DAB.  All my samples come out great!  There was need for some adjustment as I was getting background initially.  But I got rid of that with time adjustment and blocking steps.  I hope this helps.


"Schmitz, Nicole" <schmitzn <@t> uni-muenster.de> wrote:
In our lab we perform RNA in situ hybridizations on paraffin sections. We use the Roche DIG Labelling Kit and the Nucleic Detection Kit (AP-conjugated antibody and NBT/BCIP staining). We tried to optimize the protocol but there are still some problems occuring. After getting rid of the background staining, which took quite a while we now have the problem that after the stopping of the final staining reaction the sections go on turning darker and darker over the next few days. Even the sense probe turns dark and if there is a high mRNA expression the sections sometimes turn so dark that you can see nothing anymore except the dark colour. Has anyone encountered that kind of problem before and has any suggestions? The problem is that we have to store the slides so just taking pictures right after staining is not the best solution..
And I have another question. While trying to solve the problem I found out that some people use an Poly d(T) oligonucleotide to measure RNA degradation in the tissue. Does anyone know more about that e.g. where to order and how to use? Would I have to use another protocol for that than my normal in situ protocol?
Thank you for your help 

Sincerely,
Nicole Schmitz
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