Anti zo 1 sc 10804

Embryos were fixed in 4% PFA, washed three times in PBS containing 0.1% Triton X-100, and incubated in 10% FBS for 1 h. Primary antibodies were incubated overnight at 4°C. Antibodies included anti-Oct4 (sc-8628; Santa Cruz Biotechnology, Inc.), anti-Cdx2 (MU392A-UC; Biogenex), anti-Yap (4912S; Cell Signaling Technology), anti-aPKC (sc-216; Santa Cruz Biotechnology, Inc.), anti–E-cadherin (U3254; Sigma-Aldrich), and anti–ZO-1 (sc-10804; Santa Cruz Biotechnology, Inc.). Embryos were washed three times in PBS/0.1% Triton X-100, and secondary antibodies coupled with Alexa Fluor 488, 568, and 647 (Jackson ImmunoResearch Laboratories, Inc.) and Draq5 were incubated at room temperature for 2 h, washed three times with PBS/0.1% Triton X-100, and analyzed at room temperature in individual microdrops of PBS/0.1% Triton X-100 on glass-bottomed microwell dishes at room temperature (19–21°C) using the confocal microscopy procedure as described in the previous section (Bessonnard et al., 2014 (link)). Images were prepared using Fiji and Gimp 2.8 software. Quantification of fluorescence was done as previously described for CFP/YFP ratiometric analyses. All experiments were repeated on more than three littermates.