[Histonet] RNA in situ hybridization-staining problems

[Jimmy Lao]

Nicole,
We used BM purple for AP-conjugated anti-DIG, I don't
have problem like you mention,  I just feel BM pruple
is very stable, For more information please visit my
working Lab website for protocal:
http://saturn.med.nyu.edu/research/dg/joynerlab/protocols.html
Hope the visualization part can help!
Jimmy

--- - - <emerald_lake77 <@t> yahoo.com> wrote:

> 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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