RNAscope (ACD) was used to perform RNA in situ hybridization according to the manufacturer’s instructions as previously described (63, 65). RNAscope probes to the following mouse genes were used for experiments: Fbln5 (ACD, 493621) and Eln (ACD, 319361). Immunofluorescent images were then captured using an automated TiE inverted fluorescence microscope and a ×100 Plain Apo objective (Nikon Instruments, Inc.).

To identify proliferating parenchymal cells, lung sections were immunostained with anti-PCNA (Cell Signaling Technology, 2586) and anti–pro-SPC (Abcam, ab90716) antibodies and imaged using a Keyence BZ-X710 fluorescent microscope with BZ-X viewer software (Keyence) with a ×40 objective. Immunofluorescent images were then imported into ImageJ for quantification of all PCNA-positive cells (all proliferating cells) and pro-SPC/PCNA dual-positive cells (proliferating type 2 alveolar epithelial cells) in the lung. Apoptotic cells were detected in FFPE lung sections using the in situ cell-death detection kit (MilliporeSigma) per the manufacturer’s suggested methods.

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