[Histonet] In situ hybridization problems

From:Anne-Sophie MARTINEZ

Dear Histonetters,
I am not sure I am supposed to tackle this kind of subject here but I 
really need help. So, let's try! It is about in situ hybridization on 
slides of adult oysters.
We have been doing experiments for ages and have met a lot of different 
problems. In particular, we struggled to have a significant signal 
(antisense probe) and no background (sense probe). After a couple of 
successfull (quite!) experiments, it stopped working, even on the same 
samples as before. Since then, we are not only unable to obtain a signal 
again, but another problem has been added: when we add the substrate of 
the enzyme (NBT/BCIP) the slides seem to "dribble" (as if they have been 
bleeched (partially) - the "blue staining" is partially in the liquid 
around the slide).
My questions are thus:
- Concerning the loosing of the signal, could it be due to the 
destruction of the RNA (by the Davidson's fixative which is apparently 
not the most appropriate, the lenght of time in paraffin (samples have 
been kept in paraffin since 2005), the block conservation at 4C, the 
number of times the blocks have been cut  ...).
- Is there some way to improve the signal although the mRNA might not be 
highly expressed or be partially damaged?
- Would somebody have had the same kind of "dribble/bleeching" problem 
as us?
Thanks a lot for your help. I run short of ideas.


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