Rat anti meca 32

For immunohistochemical analysis, left lungs stored in 100% methanol were subjected to peroxidase deactivation by incubation in 5% H₂O₂/methanol for 15 minutes at room temperature and then sequentially rehydrated in 75%, 50%, and 25% and 0% methanol in PBS. A vibratome was used to cut the rehydrated lung into 150 μm thick sections, which were incubated in IHC blocking buffer (5% goat serum in 0.5% Triton X-100/PBS [PBS‑T]) at 4°C overnight and then stained with primary antibodies in IHC blocking buffer for 3 days at 4°C. Subsequently, sections were washed 3 times in PBS‑T, incubated in secondary antibodies in IHC blocking buffer overnight at 4°C, washed 5 times in PBS‑T, mounted on slides with Dako mounting medium, and stored at 4°C. Primary antibodies used were rat anti–MECA-32 (1:15, Developmental Studies Hybridoma Bank), rat anti–CD31-FITC clone MEC13.3 (1:250, 561813, BD Biosciences), mouse anti–CD64-APC clone X54-5/7.1 (1:250, 139306, Biolegend), rat anti–CD68-APC clone FA-11 (1:50, 130-102-585, Miltenyi Biotec), rat anti-aquaporin1 (1:100, ab15080, Abcam), and mouse anti–SMA‑Cy3 clone 1A4 (1:250, A2547, MilliporeSigma). Secondary antibody used was Alexa Fluor 488 anti-rat (1:250, catalog A-11006, Invitrogen, Thermo Fisher Scientific). Nuclei were stained with DAPI (1:500).