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 4°C, 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
Thanks a lot for your help. I run short of ideas.
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