For histological staining, mice were sacrificed, and lung inflation fixation was immediately performed. After exposure of the trachea and lungs, the trachea was cannulated with a scalp vein cannula (EXELINT, Redondo Beach, CA), and 10% neutral buffered formalin was gravity perfused into the lung at a height of 25 cm. After infusion, the lung was harvested and placed in formalin for 24 hr. Whole lung was then dehydrated in 70% ethanol and embedded in paraffin. For morphologic evaluation, 5 µm thick sections were cut from the paraffin blocks and stained with hematoxylin and eosin.

Immunofluorescence staining on 10% formalin-fixed mouse lung was performed on 5 µm thick paraffin-embedded tissue sections. Tissue slides were melted at 60°C for 2 hr, following rehydration through xylene and alcohol, and finally in PBS. Antigen retrieval was performed in 0.1 M citrate buffer (pH 6.0) by microwaving. Slides were blocked for 2 hr at room temperature using 4% normal donkey serum in PBS containing 0.2% Triton X-100 and then incubated with primary antibodies diluted in blocking buffer for approximately 16 hr at 4°C. Primary antibodies included ACTA2 (1:2000, Sigma–Aldrich; RRID:AB_262054), EMCN (1:200, R and D Systems; RRID:AB_2100035), FOXFI (1:100, R and D Systems; RRID:AB_2105588), HOPX (1:100, Santa Cruz Biotechnology; RRID:AB_2687966), MKI67 (1:100, BD Biosciences; RRID:AB_396287), POSTN (1:100, ABCAM), Pro-SFTPC (1:1000, Seven Hills Bioreagents; RRID:AB_2335890), SOX9 (1:100, Millipore; RRID:AB_2239761), and LYVE1 (1:100, ABCAM; RRID:AB_301509). Secondary antibodies conjugated to Alexa Fluor 488, Alexa Fluor 568, or Alexa Fluor 633 were used at a dilution of 1:200 in blocking buffer for 1 hr at room temperature. Nuclei were counterstained with DAPI (1 μg/ml) (ThermoFisher). Sections were mounted using ProLong Gold (ThermoFisher) mounting medium and coverslipped. Tissue sections were then imaged on an inverted Nikon A1R confocal microscope using a NA 1.27 objective using a 1.2 AU pinhole. Maximum-intensity projections of multi-labeled Z-stack images were generated using Nikon NIS-Elements software (RRID:SCR_014329).

Heterogeneous cell populations from paraffin-embedded immunofluorescent confocal images (60× magnification) were characterized in Imaris (Bitplane; RRID:SCR_007370), version 9.6. An average of 185 cells from control mice and 301 cells from IH-treated mice (n = 4 mice, six images per mouse) were counted specifically for proliferating cells (MKI67), endothelial cells (FOXF1), and alveolar type II cells (SFTPC) using spot detection or manual counting of cells. The cell percentage endothelial cells was calculated as the ratio between cells stained with FOXF1 and totally counted nucleus stained with DAPI in each image. Similarly, we got the cell percentages of AT2 and proliferating cells. All the remaining cells without FOXF1, SFTPC, and MKI67 staining or a cell stained with both MIK67 and SFTPC were taken as one composition group in the composition difference analysis. For testing the composition difference of endothelial, AT2, and proliferating cells in mouse lung between experimental and control groups, a centered log ratio transformation was performed on the percentage of each cell type before applying the t-test (two tailed). The statistical p-value from the t-test was adjusted with the BH method.

Alveolar wall thickness was quantified in Imaris (Bitplane), version 9.6, using the measurement tool for autofluorescence to define the alveolar walls. A total of 20 alveolar wall thickness measurements were taken for each 60× magnification confocal image (n = 2 mice per treatment, six images per mouse). Alveolar area and MaxFeret90 area were quantified in Nikon Elements, version 4.5. Maxferet90 is the distance measured orthogonally to the maximum feret chord. The larger Maxferet90 value indicates more openness in the alveolus. General analysis was run on maximum-intensity projection confocal images (20× magnification), n = 2 mice for control- and IH-treated mice, with three images per mouse. Autofluorescence with the TRITC channel was used to define alveolar areas. Thresholding was turned on, and binary processing was created for inverting, removing objects touching borders, and filtering an object area. Airways, arteries, veins, and alveolar ducts were filtered out using a minimum value of 50 and a maximum value of 4000 using the filter on object area binary. Feature area and MaxFeret90 values were exported using Microsoft Excel.

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