Rabbit anti α sma

Frozen sections (5 μm) were fixed with acetone for 10 min, penetrated by 10% Triton for 10 min, and then blocked with 5% goat serum for 1 h. The tissues were incubated overnight with primary antibodies. Rabbit anti-fibronectin (1:200, Abcam), rabbit anti-collagen I (1:200, Abcam), mouse anti-α smooth muscle actin (1:200, Sigma), mouseanti-CD45 (1:100, BD), rabbit anti-α-SMA (1:100, Abcam), rat anti-F4/80 (1:100, Bio-Rad), rabbit anti-α-SMA (1:100, Abcam), rat anti-CD206 (1:100, Bio-Rad), and rabbit anti-α-SMA (1:100, Abcam) at 4 °C. The secondary antibodies of Alexa Fluor 488 goat anti-rabbit (1:400, Abcam), Alexa Fluor 647 goat anti-mouse (1:400, Abcam) and Alexa Fluor 647 goat anti-rat (1:400, Abcam) we reselected to incubate at room temperature for 1 h. Dye the nucleus with DAPI. The images were taken by fluorescence microscope equipped with digital camera (Olympus, Japan) orconfocal laser scanning microscope (Zwiss, Germany). Measurement of the fluorescence staining area was performed using ImageJ software.

Double immunofluorescence staining was performed to detect the colocalization of Lin28a and α‐SMA, a marker of VSMCs in tissue, and the colocalization of CD31 and α‐SMA in tissue, respectively. The tissue sections were incubated with mouse polyclonal anti‑Lin28a (1:200, Santa Cruz Biotechnologies) and rabbit anti‐α‐SMA (1:200, Abcam) antibody overnight at 4°C. Additionally, mouse polyclonal anti‑CD31 (1:200, Abcam) and rabbit anti‐α‐SMA (1:200, Abcam) antibody were used overnight at 4°C. After rinsing by PBS for three times, immunoreactivity products were visualized by incubation with appropriate Alexa Fluor 488‐conjugated secondary antibodies (1:50; Invitrogen), 594‐conjugated secondary antibodies (1:150; Invitrogen), along with DAPI (Solarbio) stain to visualize the nuclei. After rinsing, specimens were examined with a fluorescence microscope (OLYMPUS FSX100).

Renal tissue and cells were harvested and lysed by RIPA lysis buffer (Wuhan Goodbio Technology, China) containing cocktail protease inhibitors (Wuhan Goodbio Technology, China) for 30 min on ice. The total protein was obtained by high-speed centrifugation at low temperatures. Protein concentrations were quantified by a BCA protein assay kit (Beyotime Institute of Biotechnology, China), and 30 µg protein was loaded, separated on 10% or 12% SDS-PAGE, and transferred to nitrocellulose membranes. Then membranes were blotted at 4 °C overnight with mouse anti-VEGF-C (Santa Cruz, USA; 1:250), rabbit anti-Prox-1 (Angiobio, USA; 1:1000), rabbit anti-LYVE-1 (Novus, USA; 1:1000), Hamster anti-Podoplanin (Angiobio, USA; 1:200), rabbit anti-Collagen1 (Novus, USA; 1:2000), rabbit anti-α-SMA (Abcam, USA; 1:4000), rabbit anti-PDGFR-β (Abcam, USA; 1:2000), mouse anti-iNOS (Santa Cruz, USA; 1:200), rabbit anti-Arginase (Santa Cruz, USA; 1:400), rabbit anti-LC3B (Sigma-Aldrich, USA; 1:1000), rabbit anti-p62 (Abcam, USA; 1:5000), mouse anti-GAPDH (Wuhan Goodbio Technology, China; 1:2000), then were incubated with HRP-conjugated anti-IgG (Jackson ImmunoResearch, USA; 1:4000), finally were detected by ECL (Pierce, USA). Image capture and analysis were conducted by Bio-RAD (USA).