Bs60084

After deparaffinization and rehydration through degraded ethanol, the slides underwent antigen retrieval in 10 mM sodium citrate buffer for 20 min. The sections were inactivated through 3% H₂O₂ for 10 min, incubated with 3% BSA to block nonspecific binding, and were incubated with primary Prolyl oligopeptidase antibody (dilution 1:300, BS60084, BIOWORLD, Dublin, OH, USA) at 4 °C overnight. After washing with phosphate-buffered saline (PBS), the secondary antibody was incubated for 30 min at room temperature, and the color was developed with 3,3-diaminobenzidine for 2 min. The sections were counterstained with hematoxylin for 30 s and sealed with neutral gum.

Total proteins were extracted using RIPA lysis buffer. Western blot was used to detect the expression of POP, MMP2, β-actin, and TGF-β1. The total protein was separated on 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and transferred to the PVDF membrane. The blot was blocked with 5% skim milk solution for 2 h at room temperature and was incubated with primary POP antibody (dilution 1: 1000, BS60084, BIOWORLD), TGF-β1 (dilution 1:500, BA2120, BOSTER, Wuhan, China), MMP2 (dilution 1:1000, BA2120, BOSTER), and β-actin (dilution 1:5000, 66009-1-Ig, Proteintech, Rosemont, IL, USA) overnight at 4 °C. After washing three times with TBST, the blot was incubated with anti-mouse IgG (dilution 1:20,000, BS12478, BIOWORLD) or anti-rabbit IgG (dilution 1:20,000, BS13278, BIOWORLD) for 1 h at room temperature. An Enhanced Easy See Western Blot Kit was employed in order to visualize the target bands, and the intensity of bands was quantified by Bio-Rad Image Laboratory software (Version 6.0). β-actin was used as an internal reference for detecting relative expression levels.