100% methanol 10 mins (at this stage, embryos can be PBS/0.2% saponin 10 mins stored at -20C for several monthes) PBS/0.2% saponin 10 mins
The embryos can be stored at 4C like this for a week or two.
PBS/0.2% saponin/5% serum (normal goat or foetal calf) 10 mins
As discussed above, some antigens are methanol sensitive, and in this case the vitelline membranes have to be removed by hand. In this case, substitute the following for 6-9 above:
6a) Remove the aqueous (lower) phase. Take some embryos in heptane with a pipette, and spread them dropwise onto a microscope slide covered with double sided sticky tape. Let the heptane evaporate, and just as it does, immerse the slide in PBS/0.2% saponin in a petri dish; do not let the embryos dry out on the slide. Using a fine tungsten needle, gently tease the embryos out of their (already cracked) vitelline membranes; this step is made much easier if the embryos are initially fixed for longer than normal (about 45 minutes). Transfer the embryos to an Eppendorf tube of PBS/0.2% saponin/5% serum, using a Gilson pipette with a yellow tip cut off to make a wide enough opening.
original text by Matthew Freeman