Goat anti oct4 antibody

Blastocysts were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature. After washing with PBS containing 10 mg/ml bovine serum albumin (BSA; Merck # 12657) (PBS/BSA), the fixed embryos were permeabilized by 15 min incubation with 0.5% Triton X-100. After blocking in PBS/BSA for 1 h at room temperature, they were incubated in a mixture of primary antibodies including rabbit anti-H3K27me3 antibody (1/500, Millipore # 07-449), goat anti-Oct4 antibody (1/500, SantaCruz # sc-8628) and mouse anti-Cdx2 antibody (1/100, BioGenex # AM392-5M) at 4°C overnight. Following three washes with PBS/BSA, the embryos were incubated with a mixture of secondary antibodies including fluorescein isothiocyanate-conjugated anti-mouse IgG (1/400, Jackson Immuno-Research), Alexa Flour 546 donkey anti-rabbit IgG (1/400, Thermo Fisher Scientific) and Alexa Flour 647 donkey anti-goat IgG (1/400, Thermo Fisher Scientific) for 1 h at room temperature. Finally, they were mounted with Vectashield with 4’,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories # H-1200). The fluorescent signals were observed using a laser-scanning confocal microscope (Zeiss LSM510) and an EM-CCD camera (Hamamatsu ImagEM). Three to five embryos were examined for each condition.

Standard immunostaining was carried out as previously reported^{13 (link)}. Primary antibodies were goat
anti-SOX17 antibody (R&D, Cat # AF1924) diluted 1: 1,000, rabbit
anti-FOXA2 antibody (Millipore, Cat # AB4125) diluted 1:500, goat
anti-FOXA2 antibody (R&D, Cat # AF2400) diluted 1:1,000, mouse
anti-HNF4α antibody (Perseus Proteomics, Cat # PP-H1415-00)
diluted 1:500, mouse anti-AAT antibody (Thermo Scientific, Cat #
MA190438) diluted 1:500, goat anti-PDX1 antibody (R&D, Cat #
AF2419) diluted 1:1,000, goat anti-OCT4 antibody (Santa Cruz Biotechnology, Cat
# sc-8629) diluted 1:500, rabbit anti-NANOG antibody (Abcam, Cat
# ab80892) diluted 1:500, mouse anti-AFP antibody (Sigma-Aldrich, Cat
# A8452) diluted 1:500, mouse anti-CK18 antibody (Abcam, Cat #
ab82254) diluted 1:500, and goat anti-albumin antibody (Bethyl, Cat #
A80-129A) diluted 1:500. Secondary antibodies were Alexa Fluor 488/555 donkey
anti-mouse or anti-rabbit or anti-goat IgG (1:1,000) (Invitrogen). Nuclei were
visualized by Hoechst (Sigma-Aldrich) staining. Images were captured using a
Nikon Eclipse TE2000-U microscope.