For staining of iPSC-derived FPps, rabbit anti-LMX1A (Abcam, Cat. No. ab31006) and mouse anti-FOXA2/ HNF-3 beta (Santa Cruz Bio., Cat. No. sc-101060) were used at 1:500 and 1:200 dilutions, respectively. Secondary antibodies were purchased from ThermoFisher: Goat Anti-Mouse Alexa Fluor 488 (Cat. No. A-10680) and Goat Anti-Rabbit Alexa Fluor 594 (Cat. No. A-11037), both at 1:500 dilution.
Plates were washed with DPBS (ThermoFisher, Cat. No. 14040133) to remove media and debris. Cells were then fixed with 4% paraformaldehyde/DPBS solution for 10 min, followed by three washes with DPBS. Cells were permeabilized with 0.1% Triton-X100 solution for 5–10 min, followed by one wash with PBS. Cells were blocked with blocking buffer containing 5% goat serum (ThermoFisher Cat. No. 16210064) in DPBS for 1 h, followed by three washes with PBS. Primary antibody was added in appropriate concentration and incubated overnight at 4°C. On the next day, all wells were washed three times with DPBS and secondary antibody was added and incubated on a rocker for 1 h at room temperature. Cells were washed with DPBS three times and a Hoechst 33342 (ThermoFisher Cat. No. H3570, 1ug/mL) nuclear stain solution was added to each well and incubated for 5–10 min. Cells were washed three times with PBS and stored at 4°C before fluorescent imaging.
Plates were washed with DPBS (ThermoFisher, Cat. No. 14040133) to remove media and debris. Cells were then fixed with 4% paraformaldehyde/DPBS solution for 10 min, followed by three washes with DPBS. Cells were permeabilized with 0.1% Triton-X100 solution for 5–10 min, followed by one wash with PBS. Cells were blocked with blocking buffer containing 5% goat serum (ThermoFisher Cat. No. 16210064) in DPBS for 1 h, followed by three washes with PBS. Primary antibody was added in appropriate concentration and incubated overnight at 4°C. On the next day, all wells were washed three times with DPBS and secondary antibody was added and incubated on a rocker for 1 h at room temperature. Cells were washed with DPBS three times and a Hoechst 33342 (ThermoFisher Cat. No. H3570, 1ug/mL) nuclear stain solution was added to each well and incubated for 5–10 min. Cells were washed three times with PBS and stored at 4°C before fluorescent imaging.