Fitc conjugated goat anti rabbit second antibody

The subcellular localization of endogenous Smad proteins including smad2 and smad3 was determined using an immunofluorescence study as previously described [12 (link), 13 (link)] using the same primary antibodies as used in the WB analysis and FITC-conjugated goat anti-rabbit second antibody (1 : 100; Abcam). Pictures were captured with a digital camera coupled with a fluorescent microscope (Olympus).

All steps were performed at room temperature unless mentioned. The collected oocytes and embryos were fixed with 4% paraformaldehyde for at least 30 min, permeated for 20 min with 0.5% Triton X-100 in PBS, and all samples were incubated in the blocking solution (2% BSA, 2% skimmed milk powder, 0.15 mmol/L glycine and 0.05% Tween-20 in PBS) for 1 h, and then they were incubated with anti-NLRP7 (GeneTex, N3C2) antibody with a dilution of 1:200 for overnight at 4°C. After three times of washing with PBS containing 0.1% Tween-20 and 0.01% Triton X-100 for 5 min, the samples were incubated with FITC conjugated goat anti-rabbit second antibody (Abcam, dilution 1:500) for 2 h at 37°C. After three times of washing with PBS containing 0.1% Tween-20 and 0.01% Triton X-100 for 5 min, the DNA was stained with DAPI (C1005, Beyotime Institute of Biotechnology). Finally, the oocytes or embryos were mounted on glass slides and were observed with a fluorescent microscope.