I am doing some staining experiments in mice embryos and I noticed that I lose the zona of pellocida during the procedures. I can not define at exactly which step I lose it but mostly either during or after fixation. my procedures include Satining with mitotracker, washing M2 media, 4% PFA fixation, washing with PBS+ 0.3% triton, staining with dapi and then washing again and mounting.
Is it normal to lose the zona of pellocida or I am doing something wrong?
You may try excluding 0.3% triton from the washing solution. For mitotracker or DAPI the embryos will become sufficiently permeable to the dye during the fixation steps. Moreover, please be very gentle while washing the embryos. Vigorous pipetting may often cause zona disruption. I hope this will help.
Best
Rahul