Hello! In parallel with your favorite gene, have you also included a positive control (eg: a probe surely working?). Which in situ protocol are you adopting? Greetings, Natascia

Ya, I did that also. Still I m getting same results. Is that due to improper blocking?

(I hav uploaded the pic with the ques.)

Hi! Well, it can be everything, the in situ is a quite long process and background can arise because of the probe, because of the hyb. temperature, because of the antibodies, because of the washes, because of the staining buffer, and so on. If you let me know what protocol you are using, I can tell you which can be the most critical steps. Greetings, Natascia

Thank you Natascia. Am following this protocol.

http://www.nature.com/nprot/journal/v3/n1/full/nprot.2007.514.html

I see; that's one of the best protocols, indeed; the same I am also following. Well, if any of the troubleshooting examples mentioned in there may apply to you, I would not know what else to suggest; maybe just try to re-synthesize and purify again the probes, wash embryos with larger volumes of solutions, pre-block the antibodies with fragmented embryos at different stages, try different hybridization temperatures... Good luck! Nat