Ab5694

Immunofluorescence staining was performed as described previously [38 (link),39 (link)]. Briefly, VSMCs were seeded in a 24-well plate with glass coverslips and subjected to different treatments. Next, VSMCs were fixed with 4% paraformaldehyde for 15 min and permeabilized for 30 min with 0.1% Triton X-100 in PBS. After washing with PBS three times, cells were blocked with 10% BSA and incubated overnight at 4 °C with Ki67 antibody (1:1000, 9449T, CST), γ-H2AX antibody (1:500, ab81299, Abcam), α-SMA antibody (1:500, ab5694, Abcam) and calponin antibody (1:500, ab5694, Abcam). Subsequently, VSMCs were washed and incubated with the fluorescent-labeled secondary antibodies (1:500, ab150116, ab150080, Abcam) for 1 h at room temperature in the dark. Finally, glass coverslips were mounted with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Inc.) to stain the nucleus. Six view fields from each coverslip were randomly captured with a fluorescent microscope.

Mice were sacrificed on indicated days. Tumor tissues were completely dissected, fixed in 4% buffered paraformaldehyde, embedded in paraffin, and were subsequently cut into 5-µm-thick sections. The sections were dewaxed and then placed in citric acid buffer (PH 6.0) for microwave antigen retrieval. Endogenous peroxidase of sections was then blocked with 3% hydrogen peroxide solution. Sections were further blocked with 3% bovine serum albumin blocking solution (Sigma) and immunostained with rabbit anti-mouse PD-L1 (ab233482), rabbit anti-mouse α-smooth muscle actin (SMA) (ab5694), rabbit anti-FN1 (ab2413), rabbit anti-CDH1 (ab76319) and rabbit anti-Ki-67 (ab15580) purchased from Abcam, UK at 4 ℃ overnight. The sections were then washed well with phosphate buffered saline with Tween-20 (PBST) and incubated with goat anti-rabbit IgG secondary antibody (ab150077, abcam) at room temperature for 1 hours. After being washed with PBST, staining was developed with DiAminoBenzidine. Slides were counterstained with hematoxylin, dehydrated, and finally mounted. These sections were observed under a microscope and were measured by using Image-pro Plus 6.0 software.

Whole-cell protein was extracted from kidneys or IMCD3 cells using Radio immunoprecipitation assay (RIPA) buffer (Thermo Scientific, USA). An equal amount of proteins (20 µg) was loaded on a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The membrane was then blocked with 5% skim milk and incubated overnight at 4 °C with anti-DJ-1 (1:1,000, 2134S, CST, USA), anti-Ki67 (1:1,000, ab16667, Abcam, USA), anti-PCNA (1:2,000, ab18197, Abcam, USA), anti-Bax (1:500, 2772S, CST, USA), anti-Bcl-2 (1:500, ab196495, Abcam, USA), anti-α-Smooth muscle actin (SMA, 1:1,000, ab5694, Abcam, USA), anti-fibronectin (FN1, 1:1,000, ab2413, Abcam, USA), anti-TGF-β1 (1:1,000, ab92486, Abcam, USA), anti-p53 (1:5,000, ab26, Abcam, USA), anti-Cytc (1:5,000, ab133504, Abcam, USA), anti-cleaved-caspase 3 (1:1,000, ab2302, Abcam, USA), and anti-cleaved-caspase 9 (1:1,000, ab2324, Abcam, USA) antibodies. Subsequently, the goat-anti-mouse Horseradish Peroxidase (HRP)-conjugated IgG antibody (1:5,000, Cat. No. ab205718, Abcam, USA) was used as a secondary antibody. Finally, bands were developed with an Enhanced Chemiluminescence Kit (Thermo Scientific, USA) and quantified by Quantity One imaging software (BioRad, USA) normalized to β-actin (internal control).