Polyclonal igg alexa fluor 488 donkey anti rabbit

Embryos for immunofluorescent detection were processed following the protocol proposed by (Ungos et al., 2003). Embryos were fixed in 4% paraformaldehyde in PBS (phosphate‐buffered saline; Oxoid, Basingstoke, Hampshire, UK) overnight at 4°C. They were washed three times for 5 min in PBTriton (0.1% Triton X‐100 Sigma‐Aldrich in PBS) and three times for 1 h in MilliQ water. Embryos were incubated in block solution (1% DMSO, 5% Goat/Sheep serum diluted in PBTriton) for 2–3 h. They were then incubated at room temperature overnight in polyclonal mouse serum primary antibody (polyclonal IgG rabbit anti‐GFP primary antibody; Invitrogen, A11122) diluted 1:500 in block solution. Embryos were washed in PBTriton once briefly and three times for 1 h. They were incubated overnight at 4°C in Alexa Fluor 488 fluorescent anti‐rabbit secondary antibody (polyclonal IgG Alexa Fluor 488 donkey anti‐rabbit; Invitrogen, A21206) diluted 1:200 in block solution. Embryos were then washed once briefly and three times for 30 min in PBTriton. They were stored in 50% glycerol for imaging and storage.