Anti lyve 1

Immunofluorescence staining was performed as described previously^{23 (link)}. Briefly, rat kidneys were fixed with 10% neutral buffered formalin, cryoprotected in 10% sucrose in PBS (6 h at 4 °C), immersed overnight in 20% sucrose in PBS at 4 °C, and frozen in OCT Tissue-Tek compound (SAKURA, Cat#: 4583) before 6 μm-thick cryosections were prepared. Anti-VEGFC (Immunoway, Cat#: YT5297), anti-CD68 (Abcam, Cat#: ab955), anti-LYVE-1 (Novus Biologicals, Cat#: NB600-1008), anti-α-SMA (Abcam, Cat#: ab202509), anti-vimentin (Abcam, Cat#: ab8978) and anti-collagen I (Abcam, Cat#: ab270993) were used to examined the frozen kidney sections. Secondary antibodies conjugated with Alexa 488 (Abcam, Cat#: ab150113, ab150081), Alexa 555 (Abcam, Cat#: ab150078, ab150106) and DyLight 405 (Abcam, Cat#: ab175651) were used to visualize antigen-antibody complexes. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Digital images were then obtained by confocal scanning microscopy (CTS SP8, Leica, Germany).

hLECs were treated with aldosterone and analyzed by flow cytometry. The cells were collected and stained with anti-LYVE-1 (Novus Biologicals, Cat#: NB600-1008), after which they were incubated with Alexa 647-labeled goat anti-rabbit IgG (Abcam, Cat#: ab150079) or PE-conjugated anti-VEGFR3 (Biolegend, Cat#: 356,204) secondary antibodies for 1 h. If intracellular staining was needed, a fixation and permeabilization procedure was used (eBioscience™ Foxp3, Invitrogen, Cat#: 00-5523-00). Then, the cells were stained with Alexa Fluor® 647-conjugated anti-α-SMA (Abcam, Cat#: ab202296), APC-conjugated anti-vimentin (Invitrogen, Cat#: MA5-28,801) and FITC-conjugated anti-Ki67 (Invitrogen, Cat: 11-5698-82) for 1 h in the dark. The cells were then analyzed on a BD Accuri C6 flow cytometer.