I have frozen sections of 4%PFA drop-fixed, 30% sucrose dehydrated fetal livers from E14.5 mouse embryos. I sectioned them both at 10 and 5 microns. When I performed the staining on the 10 micron sections the DAPI was extremely dense and therefore I went thinner on the next round. However in both attempts I could not detect any c-Kit signal despite confirming the antibody does work on whole embryo sections. I am using c-Kit eBioscience cat. 14-1171-82

My questions are:

1. Is it just a matter of hit or miss? To my understanding there is not a robust amount present in the fetal liver at any point in time so maybe it is just a matter of staining enough slides to catch some c-kit positive cells?

2. Is there something extra needed to be done as far as the tissue prep? Most protocols in the lit are very vague. The fetal liver is full of blood cells and maybe I should consider perfusing, but then is it possible I also end up flushing out my cells of interest (HSC)?

Thanks in advance!