Rabbit anti collagen 1

Cultured cells were fixed with 4% paraformaldehyde for 10 min at room temperature. Mouse corneal cryosections were fixed with ice methanol for 10 min at −20 °C. All samples were blocked with 5% BSA (Sigma-Aldrich Corp, St. Louis, MO, USA) and incubated with FITC-conjugated phalloidin (Alexis Biochemicals, San Diego, CA, USA), rabbit anti-α-SMA (Abcam), rabbit anti-Collagen I (Abcam) and rabbit anti-Nrf2 (Abcam) antibodies at 4 °C overnight. The samples were subsequently incubated with Alexa Fluor 488-labeled donkey anti-rabbit IgG antibody (Invitrogen, Carlsbad, CA, USA) at 37 °C for 1 h. Nuclear counterstaining was performed using 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). Finally, the staining was observed under a confocal laser-scanning microscope (Nikon, Tokyo, Japan) or a fluorescence microscope (Nikon).

Collected skin sections were fixed at a 10% buffered formalin solution at 4 °C for 24 h, dehydrated in isopropyl alcohol (70%, 80%, 96%, and 100%), embedded into the paraffin, and cut on a thickness of 5 μm using a rotating microtome (Leica, Wetzlar, Germany). All slides were stained with hematoxylin-eosin (H&E) and the following immunohistochemical markers: rabbit anti-MMP9 in a 1:25 dilution (Lab Vision; Thermo Scientific, Rockford, IL, USA); rabbit anti-collagen I in a 1:300 dilution (Abcam; Cambridge, UK); rabbit anti-CD34 in a 1:3500 dilution (Abcam; Cambridge, UK); and rabbit anti-Iba1 in a 1:8000 dilution (Abcam; Cambridge, UK). For visualization, we used Mouse and Rabbit EnVision Detection Systems, Peroxidase/DAB detection IHC kit (DAKO Agilent; Manchester, UK). Before the antibody application, we performed a retrieval reaction. For anti-collagen I and anti-MMP9, the TRIS base buffer (pH 8.2) was used, and for anti-CD34 and anti-Iba1, the citrate buffer (pH 6.0) was used. Antibodies were applied for 60 min at room temperature with Mayer’s hematoxylin counterstain and finally mounted with a DPX medium (Sigma–Aldrich, Darmstadt, Germany). An analysis of slides was performed under a Leica DMLB 100T professional biological microscope (Leica, Germany), and slides were scanned using a digital microscope VisionTek^® (Sakura, Osaka, Japan).

Rat glomerular mesangial cells (RMC, HBZY-1, Life-Science Academy of Wuhan University, Wuhan, China) were cultured and maintained in DMEM (Invitrogen, Carlsbad, CA), PH7.4, supplemented with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA), 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin at 37 °C. To examine the effect of compound 1, RMC cells were pre-treated with indicated concentration of compound 1 at 37 °C for 1 h, and then exposed to either 5.6 mM (normal glucose, NG) or 25 mM (high glucose, HG) D-glucose for 24 h or 48 h. Whole cell proteins were extracted using a cell lysis buffer kit (Cell signaling, MA, USA) and quantified by the Bradford assay (Bio-Rad, Hercules, CA). The equivalent amount of proteins were resolved with SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked and then incubating with a rabbit anti-fibronectin pAb (Sigma, MO, USA), rabbit anti-collagen I (Abcam, MA, USA), rabbit anti-p47phox, rabbit anti-Nox4, rabbit anti-PCNA, rabbit anti-c-myc (all from Santa Cruz, Texas, USA) and mouse anti-α-tubulin (Sigma) at 4 °C overnight. After washing, the membranes were incubated with HRP-conjugated anti-rabbit or anti-mouse IgG secondary antibodies and detected by using ECL detection system.

KLNs were frozen and sectioned with a cryomicrotome. After blocking with 3% (vol/vol) bovine serum albumin in phosphate-buffered saline (PBS) for nonspecific antigens, sections were incubated overnight with primary antibodies at 4°C, followed by secondary conjugated antibodies for 1 hour at room temperature. The following antibodies were used: rat anti-MECA79 (Novus Biologicals, 1:200), goat anti-PDPN (R&D Systems, 1:200), rabbit anti-fibronectin (Abcam, 1:300), rabbit anti-collagen I (Abcam,1:300), rabbit anti-LYVE-1 (Abcam, 1:300), fluorescein (FITC)-conjugated rat anti-LYVE-1 (BioLegend, 1:100), and rat anti-TGFβR1 (Santa Cruz, 1:200). The secondary antibodies used were either FITC-anti-rat, Cy3-anti-rabbit/goat, or aminomethylcoumarin acetate (AMCA)-anti-goat (Jackson ImmunoResearch, 1:200). Images were captured using a EvosFL Auto2 fluorescence microscope. Areas of positive staining were assessed semi-quantitatively by ImageJ software (NIH). Statistical analysis was performed using two-tailed Student’s t tests by Prism (GraphPad Software, Inc).