Cts sp8

The cells were cultured on 24-well chamber slides and fixed with 4% paraformaldehyde for 20 min at 4 °C. After 0.25% Triton X-100 was added and incubated for 15 min for permeabilization, the cells were incubated with antibodies against CD34, α-SMA, F4/80, and VEGFA overnight at 4 °C. Then, the cells were incubated with TRITC/FITC-labeled secondary antibodies (1:200) for 1 h at 37 °C and the nuclei were stained with DAPI for 10 min. Images were captured using a confocal microscope (CTS SP8, Leica, Weztlar, Germany) and EVOS^® FL Auto (Thermo Fisher, Waltham, MA, USA).

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).

For fluorescence staining, kidneys were irrigated with 4% PFA, dehydrated in 30% sucrose, and frozen in OCT compound (Sakura, Torrance, CA, USA). 6µm kidney sections were cut using a freezing microtome and prepared for staining with Alexa Fluor 555-conjugated α-SMA (1:500, Abcam, Cat#: ab202509) or the following unconjugated antibodies: anti-collagen I (1:50, Abcam, Cat#: ab270993), anti-Ki-67 (1:100, Abcam, Cat#: ab15580), anti-F4/80 (1:200, Abcam, Cat#: ab186073), anti-iNOS (1:50, Novus, Cat#: NB300-605), and anti-CD206 (1:50, Abcam, Cat#: ab64693). Then the sections were subjected to second or third fluorescence staining. After being stained, sections were incubated with or without DAPI for nuclear staining and sealed for photography using a confocal microscope (CTS SP8, Leica, Germany).