Q5 camera

Formalin‐fixed, paraffin‐embedded kidney tissue sections were deparaffinized, rehydrated, and permeabilized with 0.1% Triton solution (BioRad). Sections were blocked with 10% Aqua Block (East Coast Bio) solution in PBS and incubated with antibodies against LEC markers Lymphatic Vessel Endothelial Hyaluronan Receptor 1 (LYVE‐1) and Podoplanin (Table 1). Following fluorescent secondary detection, slides were mounted with ProLong Gold antifade reagent containing 4′,6‐diamidino‐2‐phenylindole (Invitrogen) and imaged using an Olympus BX51 fluorescence microscope with an Olympus Q5 camera. Representative images were captured at 10x magnification using Olympus CellSens imaging software (Olympus). All LYVE‐1+, lumen‐containing lymphatic vessels found around interlobular arteries at the cortex and corticomedullary junction, excluding hilar vessels, by two independent, blinded investigators. For quantification of lymphatics based on podoplanin expression at different time points, five images from predetermined areas within the renal cortex of each kidney section were captured at 10× magnification, with efforts made to exclude tissue defects and glomeruli from the images. The area values measuring the total number of podoplanin + pixels on the images (with the glomerular area removed) were determined using ImageJ (NIH) after setting the threshold for positive endothelium.

At euthanization, kidneys were collected, cut sagittally into halves, and fixed in 10% buffered formalin solution (Sigma, St. Louis, MO, USA) for 48 h. Following fixation, kidney halves were washed in 100% ethanol and embedded in paraffin. Kidney halves were cut into 5–7 μm sections, which were deparaffinized, rehydrated, and permeabilized with 0.1% Triton solution (BioRad, Hercules, CA, USA). Tissue was blocked with 10% AquaBlock solution (EastCoastBio, North Berwick, ME, USA) before being immunolabeled with the lymphatic endothelial cell markers LYVE-1 or podoplanin (goat polyclonal; R&D Systems, Minneapolis, MN, USA) by overnight incubation at 4 °C. Alexafluor 488 or 594 (Life Technologies, Carlsbad, CA, USA) secondary antibodies were used to visualize lymphatic vessels. Samples were incubated with appropriately conjugated secondary antibodies for an hour at room temperature. Negative controls were incubated with only a secondary antibody. All labeled slides were mounted with ProLong Gold anti-fade reagent containing DAPI (Invitrogen, Carlsbad, CA, USA). An Olympus BX51 fluorescence microscopy system with Olympus Q5 camera was used for imaging. Images were captured at 40× magnification using Olympus CellSens imaging software (Olympus, Shinjuku, Tokyo, Japan). The same methods were utilized to image lymphatics in the heart, liver, lung, and skin (10× or 20× magnification).