I am trying to elucidate the expression patterns of a number of candidate genes in deeper tissues (i.e. the visceral mesoderm and gut) of stage 9-13 embryos, and am currently employing the protocol outlined by Kosman et al. (http://www.sciencemag.org/content/305/5685/846.abstract) for RNA probe generation and subsequent fragmentation, purification, as well as embryo prep, incubation, and immunostaining. So far I have had inconsistent results, which I suspect to be due to inadequate RNA probe concentration and improper penetration of riboprobes into deeper tissues. I have not had any issues with FISH when assaying for genes expressed in the ectoderm, and have found variability when assaying for genes with known expression patterns in deeper tissues (i.e. within the same batch of samples and conditions, the same riboprobe shows a decent expression pattern in some samples and not in others).

Does anyone have any tips for improving riboprobe concentration and permeabilization of tissues? I have used DNA concentrations anywhere between 200ng to 1ug, followed sterile technique to the best of my ability, and varied probe incubation times anywhere between 16-24 hours at temperatures ranging from 56C to 64C. I have also used Proteinase K digestion at the recommended concentration (10ug/mL) for 6 minutes. Any suggestions would be greatly appreciated -- thank you very much in advance.