Sc 7705

To assess the evaluation of Nox4 deletion in AT2 cells, the lung tissue sections from Nox4^fl/fl and Nox4^−/−Spc-Cre mice (Nox4 deleted) were pretreated with antigen retrieval buffer (10 mM citrate), followed by blocking nonspecific binding with appropriate serum. Sections were then subjected to immunofluorescence co-staining for NOX4 (1:300; 14347-1-AP; Proteintech, Rosemont, IL, United States) and surfactant protein-C/ SPC (1:50; sc-7705; Santacruz, Dallas, TX, United States) to co-localize NOX4 deletion in AT2 cells. This was followed by incubation with respective secondary antibodies (Alexa Fluor 488 and Alexa Fluor 594 from Invitrogen at 1:500) followed by mounting using Vectashield mounting medium containing 4’,6-diamidino-2-phenylindole/DAPI (Vector Laboratories, Burlingame, CA, United States). Appropriate negative controls were run by omitting the primary antibody to confirm nonspecific staining. Images of the immunostained sections were captured using a fluorescence microscope (Leica Microsystems, Wetzlar, Germany) equipped with a Rolera XR CCD camera (Q-Imaging, Surrey, BC, Canada) mounted on a microscope. ImageJ software was used to analyze the stained lung sections and the intensity of staining was measured per area.

The trachea was cannulated, and the lungs were perfused with 10% neutral-buffered formalin. The left lung was removed, post fixed for 2 days at 4 °C, paraffin-embedded, and sectioned (5 μm). Sections were deparaffinized, pretreated for antigen retrieval, and incubated with antibodies against surfactant protein C (SPC) (1:50; sc-7705, Santacruz, Dallas, TX, USA) and NOX4 (1:300; 14347-1-AP, Proteintech, Rosemont, IL, USA) overnight at 4 °C, followed by respective secondary antibodies (Alexa Fluor 488 and Alexa Fluor 594 from Invitrogen at 1:500). Appropriate negative controls were run by omitting the primary antibody to confirm nonspecific staining. The immunostained sections were cover-slipped with a Vectashield mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA) and visualized with a fluorescence microscope. Images of the immunostained sections were captured with a Rolera XR CCD camera (Q-Imaging, Surrey, BC, Canada) mounted on a microscope (Leica Microsystems, Wetzlar, Germany). Images were taken at 80× magnification and NOX4 expression (green) in SPC-stained (red) cells (yellow merged) was quantified.

IF and IHC was performed overnight keeping the slides in humified chambers^{11 (link)} and incubating them with the following primary antibodies: ΔNp63 (ab172731, Abcam, 1:500), GFP (ab13970, Abcam, 1:200), Ki67 (ab15580, Abcam, 1:1,000), cytokeratin 5 (ab53121, Abcam, 1:250), acetylated tubulin (T7451, Sigma-Aldrich, 1:200), mucin 5ac (MA1-21907, Thermo Fisher Scientific, 1:100), cleaved caspase 3 (9661, Cell Signaling, 1:200), CC10 (sc-25555, Santa Cruz, 1:200), SPC (sc-7705, Santa Cruz, 1:100) and NGFR (ab8874, Abcam, 1:100).