[Histonet] In Situ on Human Alpha tubulin (RNA biotinylated probe)

[Reuel Cornelia]

Hi, 
Can somebody help me with ISH? I have a lots of background staining
(precipitation) on my negative control. I'm using Dako TSA GEN Point. If
anyone who have the best of their knowledge can tell me how to eliminate
this by experience, please help me. I'm using Human Alpha tubulin (RNA
biotinylated probe)(Maxim biotech) applied to goat muscle and human
muscle. I did antigen retrieval,proteinase K, Antigen Retreival/pepsin
pre treatment, H2O2 in methanol for 10 mins., denatured  at 95 C for 5 
mins, hybridized for 20 hrs/ overnight at 37C,stringent wash for 20 mins
at 55 C and then proceed with Dako TSA kit.Wash with TBSTween. Both
negative muscle have background stain that looks like the reaction we
have seen on our positive control although it is not as intense with our
positive. 
I also  used ChromaGene from immunovision but the reaction is so weak.

Question?
1. Would this be because of my tissue?
Human muscle fix in STF, goat muscle fix in paraformaldehyde. Goat
muscle gives lesser background and cleaner but still there is a
background that looks like my positive control. I wanted to make it
clean.
2. Would my probe have something to do with it.
3. How about my stringent wash? 
I know  there are so many factors and i'm looking for anything that
help me answer my questions and my help will be from your expertise.

i'm looking forward for your reply. Thank you. 

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