Mouse anti gata4

For whole-mount immunofluorescence (IF) analysis, isolated embryos were fixed in 4% paraformaldehyde in PBS for 20 min at room temperature, washed in 2% bovine serum albumin (BSA)/PBS and incubated in the permeabilization solution (0.5% Triton X/1.0% BSA/PBS) for 20 min at room temperature. After washing twice in 2% BSA/PBS, embryos were incubated with primary antibodies in 2% BSA/PBS overnight at 4%, washed three times with 2% BSA/PBS, incubated with secondary antibodies and 4,6-diamidino-2-phenylindole (DAPI) in 2% BSA/PBS for 1 h at room temperature, washed three times with 2% BSA/PBS and mounted in VECTASHIELD Mounting Medium [Vector Laboratories, (H-1000)]. The primary antibodies used were as follows: anti-mouse NANOG [rat monoclonal; eBioscience, (eBio14-5761)], anti-mouse POU5F1 [mouse monoclonal; Santa Cruz, (sc-5279)], anti-mouse GATA4 [goat polyclonal; Santa Cruz, (sc-1237)], anti-mouse CDX2 [rabbit monoclonal, clone EPR2764Y; Abcam, (ab76541)]. The secondary antibodies used were as follows: Alexa Fluor 488 anti-rat IgG [Life Technologies (A21208)], Alexa Fluor 555 anti-rabbit [Life Technologies (A31572)], Alexa Fluor 568 anti-mouse IgG [Life Technologies (A10037)] and Alexa Fluor 647 anti-goat IgG [Life Technologies (A21447)] (all donkey polyclonal). Image data were obtained and processed by a confocal microscope [Olympus, (FV1000)].

ChIP assays were performed by using a standard protocol (Millipore) with mouse anti-GATA4 (Santa Cruz, sc-25310 X), control mouse IgG, rabbit anti-SMAD2 (Cell Signaling, 5339 S), or control rabbit IgG. Eluted DNAs were analyzed by qPCR. The primer sequences are provided in Supplementary Table S3.