Rat anti cd45

Primary antibodies: Rabbit anti-cleaved Caspase 3 (9661S, Cell Signaling Technology, Danvers, USA), rabbit anti-Ki-67 (ab16667, Abcam, Cambridge, UK), rat anti-CD31 (DM3614P, Dianova, Hamburg, Germany), goat anti-CD32b (AF1460, R&D Systems, Minneapolis, USA), goat anti-Lyve1 (AF2125, R&D Systems, Minneapolis, USA), rat anti-Endomucin (14–5851–82, Thermo Fisher Scientific, Waltham, USA), rabbit anti-TRP-2 (ab74073, Abcam, Cambridge, UK), rabbit anti-wide spectrum cytokeratin (ab9377, Abcam, Cambridge, UK), goat anti-alpha smooth muscle actin (ab21027, abcam, Cambridge, UK), rat anti-CD45 (550539, BD Biosciences, Franklin Lakes, USA), rat anti-CD68 (137002, BioLegend, San Diego, USA), rabbit anti-Melan A (NBP1-30151, Novus, Minneapolis, USA). For Western blotting: rabbit anti-Akt (9272S, Cell Signaling Technology, Danvers, USA), rabbit anti-phospho-Akt (9271S, Cell Signaling Technology, Danvers, USA). Secondary antibodies: Alexa-Fluor 488, Alexa-Fluor 647 and Cy3-conjugated secondary antibodies were purchased from Dianova (Hamburg, Germany). For Western Blotting a rabbit anti-IgG HRP conjugated antibody was used. (Merck, Darmstadt, Germany).

Mice were euthanised, and dissected corneas were fixed in 100% methanol for 1 h at 4 °C and then washed in PBS. Corneal flat mounts were incubated in 20 mM ethylenediaminetetraacetic acid for 60 min at 37 °C and blocked with 3% bovine serum and 0.3% Triton X-100 in PBS for 60 min at room temperature. For immunostaining, tissues were incubated overnight at 4 °C with primary antibody rabbit anti-β tubulin III (1:500; #T2200, Sigma, St Louis, MO, USA) and rat anti-CD45 (1:500; #550539, BD Biosciences, Franklin Lakes, NJ, USA) or rabbit anti-Iba1 (1:500; #019-19741, Wako, Osaka, Japan) and rat anti-Ki67 (1:500; #14-5698-80, eBiosciences, Carlsbad, CA, USA). Afterwards, tissue flat mounts were washed with PBS before incubation with the secondary antibodies, goat anti-rabbit Alexa Fluor 647 (1:500; #A21244, ThermoFisher Scientific, Carlsbad, CA, USA) and goat anti-rat Alexa Cy3 (1:500; #A10522, ThermoFisher Scientific, Carlsbad, CA, USA) and Hoechst (1:1000; Sigma, St Louis, MO, USA) for 120 min at room temperature. Immunostained samples were then washed and mounted onto glass slides with aqueous mounting medium and coverslipped for imaging.

Paraffin sections were stained with antibodies against fibronectin, collagen I, and α-SMA. After fixation and antigen retrieval, nonspecific binding was blocked with protein block (Dako, Carpinteria, CA). Kidney sections were then incubated with rabbit anti-collagen I antibody (Rockland Immunochemicals, Gilbertsville, PA), rabbit anti-fibronectin antibody (Sigma-Aldrich, St. Louis, MO), or rabbit anti-α-SMA antibody (Abcam, Cambridge, MA) followed by Alexa-488 conjugated donkey anti-rabbit antibody (Invitrogen, Carlsbad, CA). For double immunofluorescence staining, renal tissues were embedded in OCT compound, snap-frozen on dry ice, cut at 5 μm thickness, and mounted. Kidney sections were fixed and stained with rat anti-CD45 (BD Biosciences) and platelet-derived growth factor receptor (PDGFR)-β (Santa Cruz Biotechnology) followed by appropriate secondary antibodies sequentially. Slides were mounted with medium containing DAPI. Fluorescence intensity was visualized using a microscope equipped with a digital camera (Nikon Instruments Inc., Melville, NY). Quantitative evaluation of sections stained with antibodies to α-SMA, collagen I and fibronectin was performed using NIS-Elements Br 3.0 software. The fluorescence positive area was calculated as a percentage of the total area as described10 (link)12 (link)21 (link).