Immunofluorescence staining of markers for different cell stages during differentiation

Marker proteins of different cell stages during the differentiation process from hiPSCs towards AT2-like cells were visualized by immunolabeling and confocal microscopy. For this purpose, the cells were washed once with 1× DPBS and subsequently fixed with 4% (v/v) paraformaldehyde solution (cat. 252549-500 ml; Sigma-Aldrich; St. Louis, MO) for 15 min at room temperature. Cells cultured on filters of 96-Transwell plates were fixed by adding 50 µL/well of 4% PFA in the apical compartment and 180 µL/well in the basal compartment. After three washing steps with 1× DPBS, the cells were permeabilized with 0.3% (v/v) Triton X-100 (cat. T8787-100 ml; Sigma- Aldrich) in 5% (w/v) Bovine Serum Albumin (BSA) (cat. A3059-100G; Sigma-Aldrich) in 1× DPBS for 60 min at room temperature. This permeabilization step was skipped for the cell membrane proteins EPCAM, CD184 (CXCR4) and CPM. Subsequently, the cells were washed three times with 1× DPBS, then incubated at 4 °C over night with the indicated primary antibodies (see Table ​Table66 of antibodies and respective dilution factors) and Hoechst 33342 (cat. H3570; Thermo Fisher Scientific; diluted 1:5000) diluted in 1% (v/v) BSA in 1× DPBS. The next day, the cells were washed three times with 1× DPBS and then incubated in the dark for 2 h at room temperature with species-specific secondary Alexa Fluor antibodies (see Table ​Table7),7), supplemented with 1% (v/v) BSA in 1× DPBS. Finally, the cells were washed three times with 1× DPBS. Membranes of Transwell plates were removed from the plastic support for ensuing mounting on a microscopic slide using the ProLong Diamond Antifade Mountant (cat. {"type":"entrez-protein","attrs":{"text":"P36961","term_id":"547831","term_text":"P36961"}}P36961, Thermo Fisher Scientific). Imaging of immunolabeled cells was performed using a LSM710 laser confocal microscope (Carl Zeiss Microscopy, Jena, Germany).

List of primary antibodies used in this study.

List of secondary antibodies used in this study.