α smooth muscle actin

Immunofluorescence was performed as previously described11 . The primary antibodies used were: E-cadherin (Cat No. 20874-1-AP, Proteintech, China) and α-smooth muscle actin (α-SMA) (Cat No. 14395-1-AP, Proteintech). Secondary antibodies were conjugated to Cy3 or FITC. Confocal laser scanning microscopy (TCS SP5; Leica, Mannheim, Germany) was used to visualize cells and sections. Scanning confocal microscopy was used to measure fluorescence intensity.

Bladder tissue was fixed in 10% formalin, embedded in paraffin wax and subsequently cut into continuous 5 μm thick sections. To evaluate rat bladder fibrosis, sections were stained using hematoxylin-eosin staining (HE) and Masson’s trichrome staining following standard protocols after dewaxing and washing. The sections were incubated with primary antibody at 4°C for immunohistochemical (IHC) staining, followed by washing, incubation with the secondary antibody coupled with HRP, and a final incubation at room temperature for an additional hour. Phospho-Smad2 (dilution 1:50, cat. AF3362, Affinity), phospho-Smad3 (dilution 1:50, cat. AF3449, Affinity), N-cadherin (dilution 1:50, cat. 13116, CST), vimentin (dilution 1:2500, cat. 60330, Proteintech), collagen type III (dilution 1:500, cat. 22734, Proteintech), E-cadherin (dilution 1:400, cat. 3195, CST), α-smooth muscle actin (dilution 1:1500, cat. 14395, Proteintech), TGF-β1 (dilution 1:200, cat. 21898, Proteintech), and CCN1 (dilution 1:100, cat. AF3362, Affinity) were used.

Immunofluorescence staining was performed as previously described [14 (link)]. In brief, after treatment with 3% H₂O₂ for 15 min and 10% normal goat serum for 30 min, sections or cells were incubated overnight at 4 °C with the following primary antibodies: TET2 (1:50, Santa Cruz Biotechnology), 5hmC (1:50, EPIGENTEK), 5mC (1:50, EPIGENTEK), α-smooth muscle actin (1:100, ProteinTech), Beclin 1 (1:100, bioworld), LC3 (1:100, ProteinTech), p62 (1:50, Santa Cruz Biotechnology), VCAM-1 (1:50, Santa Cruz Biotechnology), ICAM-1 (1:50, Santa Cruz Biotechnology), MCP-1 (1:200, Santa Cruz Biotechnology), and IL-1β (1:200, Santa Cruz Biotechnology). Sections or cells were then incubated with cy3-conjugated secondary antibody for 1 h at room temperature. Thereafter, the samples were counterstained with 4′,6-diamidino-2-phenylindol (DAPI) for the nuclei, followed by recording with a fluorescence microscope (IX70; Olympus, Tokyo).