Bzx700 microscopy system

Paraformaldehyde-fixed cells were washed in 0.05% v Brij-35 in PBS (pH 7.4) and processed for TUNEL labelling as per manufacturer’s instruction using TACS•XL® In-Situ Apoptosis Detection Kit (Trevigen, Gaithersburg, MD). The TdT-labelled cells were detected using fluorescently-conjugated secondary antibodies (Jackson ImmunoResearch Lab Inc., West Grove, PA). The cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) containing Fluormount-G (SouthernBiotech, Birmingham, AL) to visualize nuclei. Images were captured with BZX700 Microscopy system (Keyence Corp., Japan) and analyzed by NIH Image J software.

Differentiated brown adipocytes were washed three times with PBS, fixed with 10% (v/v) neutral buffered formalin for 10 min, washed two times with PBS, treated with 60% isopropanol, and incubated with filtered Oil red O (0.18% Oil red O in 60% isopropanol; Sigma-Aldrich) for 20 min. After washing with 60% isopropanol and subsequent PBS washes, lipid droplet images were captured with a BZX-700 microscopy system (Keyence). Stained Oil red O was re-extracted in 100% isopropanol for 5 min, and lipid accumulation was semi-quantitatively measured by the OD492. Background control comprised 100% isopropanol.

For immunohistochemical (IHC) staining, deparaffinized and hydrated lung and placental tissue sections were washed in 0.05% v Brij-35 in PBS (pH 7.4) and immunostained for antigen expression as described previously (41 (link)). Briefly, the antigens were unmasked by steaming the sections in 10 mM Citrate buffer (pH 6.0) followed by incubation in a blocking solution containing 3% BSA, 1% Gelatin and 1% normal donkey serum with 0.1% Triton X-100 and 0.1% Saponin. Serial sections were stained with antibodies to Vimentin, E-cadherin, and ZO-1 (Invitrogen Inc., Carlsbad, CA), or isotype control antibodies. The immunolabelled tissues were detected using respective secondary antibodies conjugated with fluorescent dyes (Jackson ImmunoResearch Lab Inc., West Grove, PA). Where indicated, the sections were stained with 4′,6-diamidino-2-phenylindole (DAPI) containing Fluormount-G (SouthernBiotech, Birmingham, AL) to visualize nuclei. Immunofluorescent images were captured with BZX700 Microscopy system (Keyence, Tokyo, Japan). Specific details are given under appropriate figure legends.

The murine and human AECs grown on Labtek-II slides (ThermoFisher Inc.) were fixed in 4% paraformaldehyde and washed in 0.05% v Brij-35 in PBS (pH 7.4) and immunostaining was performed as described previously¹⁴ . Briefly, the cells were blocked using a solution containing 3% BSA, 1% Gelatin and 1% normal donkey serum with 0.1% Triton X-100 and 0.1% Saponin and were stained with antibodies to MUC5AC (Millipore Inc., Burlington, MA), Spdef (Santa Cruz Biotech, Dallas, TX), Bcl-2 (Santa Cruz Biotech, Dallas, TX), Bik (Abcam, Cambridge, MA) and cleaved caspase 3 (Cell Signaling Tech., Danvers, MA) or isotype controls. The immunolabelled cells were detected using respective secondary antibodies conjugated fluorescent dyes (Jackson ImmunoResearch Lab Inc., West Grove, PA) and mounted with 4′,6-diamidino-2-phenylindole (DAPI) containing Fluormount-G^TM (SouthernBiotech, Birmingham, AL) for nuclear staining. Immunofluorescent images were captured using BZX700 Microscopy system (Keyence Corp., Japan) and analyzed using NIH Image J software.