Anti gata3

Cells and hydrogels were fixed with ice-cold 4% paraformaldehyde and incubated at room temperature for 20 min followed by three washing steps with PBS for 10 min each in room temperature. The DNA denaturation step for BrdU-treated samples was performed by incubating the samples with 2 M HCl (Fluka) for 10 min at 37°C followed by three washing steps with PBS in room temperature. The samples were permeabilized in blocking solution (0.3% Triton X-100, 10% normal goat serum, 1% BSA in PBS) for 20 min at room temperature. Samples were incubated with the listed primary antibodies in blocking solution overnight at 4°C. Primary antibodies: anti-GFAP (1:1000, Abcam), anti-SOX2 (1:300, Santa Cruz), anti-TUBB3-160 (1:500, Life Technologies), anti-GATA3 (1:300, R&D), anti-BrdU (1:500, Bio-Rad), anti-GFP (1:500, Abcam). Secondary antibodies (Alexa Fluor, Invitrogen) were used 1:500 dilution in blocking solution at room temperature for 4 h. The cells were counterstained with DAPI (1:3000 for 10 min at room temperature, Invitrogen).

At specified time points, cell cultures were 4% paraformaldehyde‐fixated (15 min/RT) followed by three 1 × PBS wash‐steps. Subsequently, cells were permeabilized for 120 min/RT within blocking solution (1 × PBS + 0.5% Triton X‐100/5% BSA). The blocking buffer was removed with no rinsing. Primary antibodies within blocking solution were then introduced/placed into incubation (overnight/4°C), with three subsequent 1 × PBS‐rinse‐steps. Fluorophore‐conjugated secondary antibodies in 3% BSA were then introduced/placed into incubation (RT/120 min/darkness). This was followed by four 1 × PBS‐wash‐steps within dark conditions. Carl Zeiss™ LSM8000® Confocal Microscopy imaging was consequently employed. Antibody specifications are listed hereunder:
Regarding human EPS cells; anti‐OCT4 (1:100, Santa Cruz™, sc‐5279); anti‐SOX2 (1:200, Abcam, ab92494); anti‐NANOG (1:200, CST, 4903S). The antibody used for formative AF9‐hPSCs was anti‐LEFTY1 (LifeSpan Biologicals, LS‐B5830). Anti‐TFAP2C (1:100, Santa Cruz, sc‐12,762), anti‐BLIMP1 (1:200, CST, 9115), and anti‐SOX17 (1:500, Neuromics, GT15094) were used for IF analysis of PGCLCs while for AF9‐induced TS‐like cells, the antibodies used were Anti‐TFAP2C (1:100, Santa Cruz, sc‐12,762), anti‐CK7 (1:200, Zsbio, ZA‐0573) and anti‐GATA3 (1:200, R&D systems, AF2605).