Rabbit anti col1a1

Kidney tissues were cut at 4 μm. Immunohistochemistry and quantification was performed according to Schinner et al. (2013) (link) Primary antibodies are mouse anti-α-SMA (Beckman Coulter, Krefeld, Germany), rabbit anti-Col1a1 and rabbit anti-fibronectin (Abcam, Cambridge, UK). Alexa 647-conjugated donkey anti-rabbit and Cy2-conjugated donkey anti-mouse served as secondary antibodies. For quantification the increase after UUO was related to the healthy kidney.

Paraffin-embedded dorsal skin sections were deparaffinized, rehydrated, and antigen was retrieved by incubating sections in boiling in sodium citrate buffer (10 mM, pH 6) for 15 min. Slides were then incubated with primary antibodies: rabbit anti-α-SMA (Abcam, Cat. ab5694, dilution 1:600) or rabbit anti-COL1A1 (Abcam, Cat. ab7778, dilution 1:600) at 4°C for 10 h. After incubation, sections were washed with 0.1 M PBS and incubated with goat anti-rabbit IgG-Alexa Fluor 488 (Abcam, Cat. ab150077, dilution 1:1000) or goat anti-rabbit IgG-Alexa Fluor 555 (Abcam, Cat. ab150078, dilution 1:1000) at room temperature for 1 h. After an additional rinse, sections were mounted by VECTASHIELD mounting medium with DAPI (Vector Lab, Shanghai, China) and observed under confocal microscopy (Olympus, Japan) (Li et al., 2021a (link)). The fluorescence intensity was measured on fields (460 μm × 460 μm) around the wound area using the ImageJ software (Li et al., 2021a (link)). At least six replicates were analyzed for each treatment.

Total RNA was extracted using Trizol (Gibco, USA), and cDNA was synthesized using Evo M-MLV RT Kit (Accurate Biotechnology, China) following manufacturers' instructions. Relative gene expression was measured relative to the endogenous gene GAPDH using the delta CT method. The ID and primer sequence of detected genes were listed (Additional file 1: Table S2).
Cell samples were lysed with RIPA buffer (Gibco, USA), and proteins were resolved by SDS-PAGE using gradient gels (4%-20%) and electroblotted onto PVDF membranes es (Millipore, German). Then membranes were probed with rabbit anti-GAPDH (1:1000; Abcam, USA), rabbit anti-α-SMA (1:1000; Abcam, USA), rabbit anti-COL1A1 (1:1000; Abcam, USA), rabbit anti-COL3A1 (1:1000; Abcam, USA) and rabbit anti-fibronectin (1:1000; Abcam, USA) at 4 °C overnight, followed by three rinses with Tris-buffered saline/Tween (TBST) and incubated at a 1:5000 dilution of goat anti-rabbit IgG secondary antibodies (CST, China). GAPDH was used as the loading control.