in situ hybridization - sense control problem - (Apr/25/2008 )

Hi

I am doing in situ hybridization with a 350bp DIG-labeled RNA probe on older (e12.5) mouse embryos. I have not been getting good results - embryos hybridized with either antisense or sense probes were stained by BCIP/NBT, even though the sense group had a bit less staining. I have tried hemisecting the embryos to increase fluid flow, and played with different permeabilization time, but the same problem still persisted. Basically I follow the protocol provided in "Manipulating the Mouse Embryos" by Nagy et al., 2003. Can someone give me some advice on how to identify the problems with my experiment and/or how to correct for them? Thanks.

i'm facing the same problem too. My get staining on both my sense and antisense probes hybridized samples, although the sense one is faint. Please someone help us out, thanks.

Hello,

I also have the problem that my in situ RNA hybridization sample sections treated either with antisense or sense probe are staining. I use plant material embedded in paraffin sections and use NBT/BCIP for detection. Increasing the stingency has not been helpfull. I know few groups working with in situ hybridization and they have faced the same problem. Someone has suggested that the blocking is not working or the secondary antibody is too old or that there can antisense inhibition be taking place in the cells (like RNAi type inhibition). So far I havent been succesfull to get rid of the problem. So if anyone has any suggestion dealing this problem, I would be thankfull.