We have tried doing genotyping PCR for some mouse embryos after complete in situ hybridization assay. But we did not get any band. I suspect something went wrong at lysis step because the positive controls have worked. Is there any particular processing needed for lysing in situ samples before genotyping?
I have never tried PCR on samples after in situ. What I end up doing, for tadpoles at least, is cut of a piece of tail for PCR while the head undergoes in situ/immuno.
Perhaps that could work better in some cases?
Thank you Bilal for the suggestion.
I thought of doing so, but it is always good to have whole embryo images. Also, in my functional studies, some probes may mark almost entire embryo.
You could use yolk sac for genotyping and keep your embryo intact.
I would also recommend to take the embryo's yolk sac during the dissection. That is common practice in my previous lab.
After the ISH you can also easily genotype the embryos, I would take a part of the embryo that does not interest you (or take the pictures before if you need to keep the whole embryo intact). Do a regular digestion followed by de-crosslinking (same tube 65°C for 1h) after the digestion and then PCR. We had no issues with the PCR afterwards.
I am harvesting embryos at very early stage. Yolk sac is not developed by that time. I tried the second part. It seems to be good. Thank you so much for the suggestion!