Af743

Mice were sacrificed with CO₂ exposure, and prior to harvest, the lungs were inflated with 4% PFA at a constant pressure of 25 cm H₂O. The lungs were fixed in 4% PFA overnight at 4°. The lung tissue was then dehydrated by immersing it in a series of ethanol solutions of increasing concentrations and embedded in paraffin for sectioning. Six-micrometer sections were immunostained with antibodies for VEGFR3 (R&D Systems, AF743) and VCAM (Abcam, ab134047). Human lung tissue was stained with antibodies for podoplanin (D240, BioLegend, 75782-960) and VCAM (Abcam, ab134047) overnight at 4°. After washing, the slides were incubated with Alexa Fluor-conjugated secondary antibodies for 1 h at room temperature. Slides were treated with DAPI-containing Vectashield that was used on the tissue sections, and a cover slip was applied. Secondary antibodies alone were used on the negative control slides to check for autofluorescence of lung tissue. Immunofluorescence was performed either using a Nikon Eclipse microscope and NIS Elements software or a Zeiss SP8 confocal microscope and Leica software. Image analysis was performed using ImageJ.