Goat anti oct4

Immunostaining assay was performed according to standard protocols. Briefly, the cells were fixed in 4% paraformaldehyde and washed with phosphate-buffered saline (PBS) (Invitrogen, 10010023) and then incubated in PBS containing 0.2% Triton X-100 (Sigma-Aldrich, 9002931) and 0.3% bovine serum albumin (Invitrogen, 11020021). Afterwards, the cells were incubated with primary antibodies including: goat anti-Oct4 (Abcam, ab27985), mouse anti-Nanog (Santa Cruz, sc-374001), mouse anti-Sox2 (Abcam, ab75485), goat anti-GATA4 (Santa Cruz, sc-1237), mouse anti-tyrosine hydroxylase (TH) (Abcam, ab6211), and goat anti- alpha fetoprotein (AFP) (Santa Cruz, sc-8108). The cells were then washed and incubated with goat anti-mouse IgG or rabbit-anti-goat IgG (Invitrogen). A concentration of 0.5 µg/ml diamino phenyl indole (DAPI) (Sigma, 28718903) was used for nuclei staining. Images were visualized using Nikon Ti inverted fluorescence microscope. Alkaline phosphatase (AP) staining was performed using the AP detection kit (Millipore, SCR004).

The cells were washed once with 1X PBS and removed with a cell scraper. The hESCs were then resuspended in SDS sample buffer (160 mM Tris 1 M pH 6.8, 4% SDS, 10% β-mercaptoethanol, 24% glycerol, and 0.02% bromophenol blue). Western blot analyses were performed with goat anti-PUM1 (1:5000, Bethyl Laboratories), rabbit anti-PUM2 (1:2500, Bethyl Laboratories), goat anti-OCT4 (1:500, Abcam) and mouse anti-β-actin (1:1000, Cell Signaling Technology) antibodies. Peroxidase-conjugated anti-goat IgG (1:2500 for anti-PUM1, 1:2000 for anti-OCT4), anti-rabbit IgG (1:2500 for anti-PUM2) and anti-mouse IgG (1:2500 for anti-β-actin) were used as secondary antibodies. HRP Chemiluminescent Substrate Reagent (Thermo Fisher Scientific) was used for chemiluminescence signal generation. The signal was captured with a ChemiExpress L-Pix instrument (Loccus Biotechnology), and the image was generated using L-Pix Image software (v. 2.11.7, Loccus Biotechnology). ImageJ software (https://imagej.nih.gov/ij/) was used for the quantitative analyses, and the gel images were quantified based on the linear signal ranges.