Mu392 uc

Immunofluorescence was performed as previously described (Dietrich and Hiiragi, 2007 (link)). The following antibodies and dilutions were used: monoclonal mouse anti-CDX2 (MU392-UC, BioGenex) 1:200, rabbit monoclonal anti-CDX2 (ab76541, Abcam) 1:200, mouse monoclonal anti-YAP (sc-101199, Santa Cruz Biotechnology) 1:200, rabbit polyclonal anti-pERM (3141, Cell Signalling) 1:250, rat monoclonal anti-E-Cadherin (U3254, Sigma) 1:250, mouse monoclonal anti-TEAD4 (ab58310, Abcam) 1:100, rabbit polyclonal anti-DsRed (632496 living colors Clontech) 1:400, goat polyclonal anti-GFP (R1091P, Acris, Origene) 1:200, rat monoclonal anti-HA (11867423001, Sigma) 1:200. Secondary Alexa Fluor conjugated antibodies (Life Technologies) were used at 1:1000. Nuclei were visualized by incubating embryos in DAPI at 1 μg/ml.

Embryos were fixed at 20℃ in 4% paraformaldehyde and 0.1% polyvinyl alcohol in PBS for 30 min, permeabilized in 0.25% Triton-X 100 in PBS for 20 min, and blocked in 3% bovine serum albumin in PBS for 1 h. Mouse monoclonal anti-CDX2 (1:500, overnight, MU392-UC, BioGenex, San Ramon, CA), rabbit polyclonal anti-OCT3/4 (1:500, o/n, sc-9081, Santa Cruz Biotechnology, Inc., Dallas, TX), and rabbit polyclonal anti-YAP1 (1:100, o/n, #14074, Cell Signaling Technology, Danvers, MA) were used as primary antibodies. Alexa Fluor–conjugated secondary antibodies (1:500; 1 h; A32728, ab175661, Abcam) were used. Laser scanning confocal images were acquired by using a CSU-W1 SoRa microscope (Yokogawa Electric Corp., Tokyo, Japan).