Mice were induced at 4 weeks with 1.5 mg tamoxifen and harvested after 7–10 days. For wholemount images, mice were sacrificed and organs were harvested. They were washed in PBS three times and images were taken on dissecting microscope using 4X magnification. For immunofluorescence analysis, sections were prepared as described previously [11 (link)]. Images were taken on Nikon TiE microscope or confocal microscope. Antibodies used are; Rabbit anti-keratin 14, polyclonal, Cat# PRB-155P, Biolegend, 1:100; Living Colors RTM DsRed (RFP) Monoclonal Antibody, Cat# 632392, Clontech, 1:100; NKCC1, Cat# sc-21545, Santa Cruz Biotechnology, 1:100; RFP, Cat# 200–301-379S, Rockland, 1:100; AQP4 antibody polyclonal, Cat# AB3594, Millipore, 1:200; Atp6v1b1 antibody, polyclonal, Cat# Pa5–56878, Invitrogen, 1:200; SCGB3A2 Antibody, Cat# AF3465, R & D Systems, 1:100; Prosurfactant Protein C (SPC) Antibody, Cat# AB3786, Millipore, 1:2000.
For kidney, tissues were fixed using published protocol [17 (link)]. Lungs were inflated with 2% paraformaldehyde in OCT and incubated for 1 hour in 3.2% paraformaldehyde in PBS. Lungs were then washed 3 × 1 hours in PBS at room temperature, followed by overnight incubation in 30% sucrose / PBS. The following day lungs were incubated 2 hours at room temperature in 15% sucrose / 50% OCT in PBS followed by embedding in OCT cryomolds.
For kidney, tissues were fixed using published protocol [17 (link)]. Lungs were inflated with 2% paraformaldehyde in OCT and incubated for 1 hour in 3.2% paraformaldehyde in PBS. Lungs were then washed 3 × 1 hours in PBS at room temperature, followed by overnight incubation in 30% sucrose / PBS. The following day lungs were incubated 2 hours at room temperature in 15% sucrose / 50% OCT in PBS followed by embedding in OCT cryomolds.