Fertilized white Leghorn chicken eggs were incubated at 38°C to reach Hamburger-Hamilton stages 11–17 (HH11–17, 40–64 hours) (Hamburger and Hamilton, 1951 (link)). Embryos were removed from eggs using a ring of filter paper adhered to the vitelline membrane and placed ventral side up into 35 mm Petri dishes, as described previously (Voronov and Taber, 2002 (link)). To eliminate artifacts due to surface tension, a second filter paper ring and a stainless steel ring were placed on the embryo to keep it submerged under approximately 1 ml of culture media. Embryos were cultured in a plastic bag with supplemented oxygen (Voronov and Taber, 2002 (link)).
To inhibit apoptosis, the irreversible caspase inhibitor Q-VD-OPh (ApexBio, A1901) or Z-VAD-fmk (ApexBio, A1902) was added to the media at stages HH11 to HH14-, before significant invagination occurred, for a concentration of 100, 150, or 200 μM. Q-VD-OPh is a pan-caspase inhibitor with very little toxicity to cells (Caserta et al., 2003 (link)), while Z-VAD-fmk preferentially inhibits caspase 3 at low concentrations (Caserta et al., 2003 (link)). Embryos were cultured overnight (16 to 23 hours), then imaged as described below.
To inhibit apoptosis, the irreversible caspase inhibitor Q-VD-OPh (ApexBio, A1901) or Z-VAD-fmk (ApexBio, A1902) was added to the media at stages HH11 to HH14-, before significant invagination occurred, for a concentration of 100, 150, or 200 μM. Q-VD-OPh is a pan-caspase inhibitor with very little toxicity to cells (Caserta et al., 2003 (link)), while Z-VAD-fmk preferentially inhibits caspase 3 at low concentrations (Caserta et al., 2003 (link)). Embryos were cultured overnight (16 to 23 hours), then imaged as described below.