mRNA in situ hybridization - help much needed (Apr/11/2005 )

Hello All,

I am trying a mRNA ISH protocol, but with no results. Tried it 3 times and couldn't get anything. I ll list the problems below.

1. The slices (brain, 30um) look very poor after the end of the protocol. Probably due to the osmolarity differences of the buffers used.(2-5X SSC)
2. There is only a high background at the end. I use DIG-labeled probes and detect with anti-DIG rhodamine Fabs (Roche)

I basically use the protocol from Liang et. al. (J. Comp. Neur. 416:475-492 (2000))

http://www.ncbi.nlm.nih.gov/entrez/query.f...t_uids=10660879

Is it that I just need to do it a few more times before it starts working, or there are some tricks to it?

Any help is very much appreciated.

Thanks to All

-Tau-

QUOTE (Tau @ Apr 12 2005, 03:40 AM)

So what is the target? HIgh expressor? That should work otherwise find one and test your method with a housekeeping gene that is highly expressed.
Why on earth 30 um sections the autoflu is huge!!!! try 10um sections.
Try TBS buffers (contains 150mm NaCl which is high enough)
Use crosslinking fixatives like PFA or FA for a short time (not ON)
And why fluorescence........ you probably won't see your signal because of the high autofluorescence of your tissue. Try DAB staining for a change.

add controls so you know at least you method works.

-FISHer-