Immunofluorescence Staining of Surfactant Proteins

Immunofluorescence staining was conducted as described [30 (link), 60 (link), 61 (link)]. Briefly, subconfluent imPAC2, imPAC2 organoids, and frozen sections of imPAC2 and imPAC2-simBC implants, were fixed with paraformaldehyde and permeabilized with 0.1% Triton X-100. The cells and sections were washed with PBS, blocked with goat serum, and incubated with primary antibodies against SftpA, SftpB, SftpD (Novus Biologicals), and/or SftpC (Abcam), followed by detection with fluorescence probes labelled secondary antibodies. Negative controls were stained without primary antibodies. Cell nuclei were counterstained with DAPI. Fluorescence images were recorded with fluorescence microscope or confocal microscopy.