Cy5 conjugated donkey anti rabbit secondary antibody

Embryos were dissected at E11.5 and fixed in 4% paraformaldehyde solution (PFA) (pH 7.4) at 4°C overnight. After the tissues were soaked in PBS/1% Triton X-100 for 20 min and rinsed with PBS several times, they were blocked with PBS (0.3% Triton X-100, 5% normal donkey serum and 1x PBS) overnight at 4°C. The tissues were incubated overnight at 4°C with primary antibodies against phospho-ERK (Cell Signaling Technology, 4,370; 1:100), phospho-AKT (Cell Signaling Technology, 8,272; 1:100), and phospho-PLCγ (Cell Signaling Technology, 8,713; 1:100). The tissues were washed 3 times with PBS for 30 min each, incubated with Cy5-conjugated donkey anti-rabbit secondary antibody (Jackson, 1:500) overnight at 4°C and washed several times with PBS. Images of stained tissue were captured using a Zeiss-LSM880 with Airyscan confocal laser scanning microscope and ZEN ImageJ software (Zeiss, Germany). The details about how the measurements were conducted on the confocal images are described in the literature (25 (link)). ImageJ was used to quantify the amount of fluorescence as mean gray value of pERK, pAKT, PLCγ and compare the results among four groups. ImageJ was also used to determine the number of stained cells by phosphorylation of histone H3 (PHH3) to compare the results among these groups.

The immunofluorescent staining of both cultured organ and in vivo organ were similar. Tissues were fixed in 4% paraformaldehyde in PBS for 20mins on ice. After soaked in PBS/1% Triton X-100 for 20 min and rinsed with PBS several times, tissues were incubated PBS with 0.3% Triton X-100 and 5% normal donkey serum overnight at 4°C for blocking. Then tissues were incubated with primary antibodies overnight at 4°C. After washing 3 times in PBS 60 min each, the tissue was incubated with secondary antibodies overnight at 4 °C, washed, and mount with 50% glycerol. Images of stained tissue were captured using LSM 710 confocal laser scanning microscope using NIH ImageJ software. The antibodies were the following: rabbit anti-SIX2 primary antibody (Proteintech, 1:200), anti-caspase3 primary antibody (Cell signaling technology, 1:100), Cy5-conjugated donkey anti-rabbit secondary antibody (Jackson; 1:500).