Mouse anti α fetoprotein afp

Cells were fixed in 4% paraformaldehyde (PFA) for 15 min, then blocked with 5% BSA prepared in PBS for 1–2 h at RT on a shaker. The main primary antibodies included mouse anti-Oct4 (Abcam, Ab19587, Shanghai, China), goat anti-SOX17 (Abcam, Ab224637, Shanghai, China), mouse anti-α-fetoprotein (AFP) (Sigma-Aldrich, A8452, Saint Louis, MO, USA), mouse anti-HNF4α (Sigma-Aldrich, SAB1412164, Saint Louis, MO, USA), and mouse anti-albumin (ALB) (Sigma-Aldrich, A6684, Saint Louis, MO, USA), which were all used at a 1:200 dilution, and incubated with cells overnight at 4 °C. After overnight incubation, cells were washed three times with PBST. Alexa Fluor 488-conjugated secondary antibodies were added and incubated with cells for 2 h at room temperature. Finally, cell nuclei were stained with DAPI (Beyotime, Jiangsu, China) and samples were visualized by means of fluorescence microscopy (Zeiss).

About 3 × 10⁶ of differentiated cells were washed once with PBS and then lysed with radio-immune precipitation assay (RIPA) buffer supplemented with a protease inhibitor cocktail (all from Beyotime, Jiangsu, China) on ice. The cell lysates were centrifuged at 12,000× g for 15 min at 4 °C, and the supernatant was collected. Protein concentration was determined using a BCA Protein Assay Kit (Beyotime, Jiangsu, China). Electrophoresis was performed in a 10% BIS-Tris gel. Proteins were then transferred to a nitrocellulose membrane and incubated overnight at 4 °C with 1:1000 dilution of primary mouse anti-α-fetoprotein (AFP) (Sigma-Aldrich, A8452, Saint Louis, MO, USA), mouse anti-albumin (ALB) (Sigma-Aldrich, A6684, Saint Louis, MO, USA), and mouse anti-β-actin (Solarbio, K200058M, Beijing, China) followed by incubation with 1:1000 dilution secondary antibody for 2 h at room temperature, and then protein bands were visualized using an enhanced chemiluminescence system. Protein expression was normalized to β-actin. All experiments were conducted at least three times. Western blotting was analyzed using Image J software, and the results expressed as the mean ± SD of the target protein/β-actin gray value.