A00090 1

Paraffin-embedded peritoneal tissues were dewaxed, rehydrated by sectioning, and blocked by peroxidase for 30 min. The tissue slices were high-pressure repaired by citrate repair solution (pH = 6.0) for 2 min. The tissue slices were sequentially incubated with primary antibodies of interest (TGF-β1[1:100], α-SMA [1:100], Smad2 [1:50], Smad3 [1:50]) overnight at 4°C. The secondary antibody (horseradish peroxidase [HRP] labelled) was incubated at 37°C for 30 min. Finally, the signal was visualized by using a 3, 3’-diaminobenzidine (DAB) kit (Beijing Zhongshan Jinqiao Biotechnology, Beijing, China). Then counterstained with hematoxylin, dehydrated with gradient alcohol and xylene and sealed with neutral balsam.
The primary antibodies used were as follows: Smad2 (A00090-1, Boster, 1:50 dilution; China); Smad3 (BA4559, Boster, 1:50 dilution; China); α-SMA (BM0002, Boster, 1:100 dilution; China); TGF-β1 (BA0290, Boster, 1:100 dilution; China).

The cells were grafted to 2.5 × 10^4/well seed containing slides in 24-well plates and cultured for 48 h. After collecting the cells, they were fixed with 4% paraformaldehyde. Cells were permeabilized with 0.2% tritonX-100 for 1h and then blocked with 5% BSA and sequentially incubated with the primary antibodies of interest and the next day after incubation using phasic secondary antibodies, The samples were washed with saline-sodium citrate (SSC) twice, stained with DAPI, and the slides were removed and sealed with an anti-fluorescence quenched tablet, and tested by fluorescence test under laser scanning confocal microscope.
The primary antibodies used were as follows: Smad2 (A00090-1, Boster, 1:200 dilution; China); Smad3 (BM3919, Boster, 1:200 dilution; China); α-SMA (BM0002, Boster, 1:200 dilution; China); TGF-β1 (MA00019, Boster, 1:200 dilution; China).