Cy2 donkey anti rabbit 1 1000 secondary antibody

Embryos were dechorionated and fixed in 2% sweet paraformaldehyde (2% PFA, 4% sucrose, and 10 mM PBS pH 7.3) overnight at 4 °C and then dehydrated and stored in 100% methanol. Proteinase-K permeabilized embryos were blocked in IB [10% FBS, 1% dimethyl sulfoxide (DMSO), and 1% Triton-X100 in DPBS] for 1 hour at room temperature and incubated with rabbit anti-GPATCH3 1:100 primary antibody (HPA032078, Sigma) and Cy2 donkey anti-rabbit 1:1000 secondary antibody (Jackson ImmunoResearch) overnight at 4 °C. Washed embryos (1% Triton X-100 in PBS) were counterstained with DAPI, oriented and mounted in Fluorescent Mounting Medium and visualized using an LSM710 Zeiss confocal microscope. Fluorescence emitted by the Cy2-conjugated antibody and embryo autofluorescence was registered at 490–518 nm and 553–677 nm respectively. Z-Stack maximum intensity projections of embryos were obtained with ZEN software (Zeiss).

Cryosections were blocked in IB (10% FBS, 1% DMSO, and 1% Triton X-100 in DPBS) for 1 hour at room temperature and incubated with rabbit anti-GPATCH3 1:100 primary antibody (HPA032078, Sigma) and Cy2 donkey anti-rabbit 1:1000 secondary antibody (Jackson ImmunoResearch) overnight at 4 °C. Then, cryosections were counterstained with DAPI, mounted in Fluorescent Mounting Medium and visualized using an LSM710 Zeiss confocal microscope. Fluorescence emitted by DAPI and the Cy2-conjugated antibody and embryo autofluorescence was registered at 411–464 nm, 490–518 nm and 553–677 nm respectively. Hematoxylin and eosin staining was performed according to standard protocols.