Anti mmp9

The endometrial tissue was mixed with lysis buffer and the protease inhibitor. Then, the samples were homogenized and centrifuged (12,000 rpm × 10 min), and the supernatants were collected. After determining the protein concentration, the samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) and transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk for 1 h at room temperature and then incubated with the primary antibody overnight at 4 °C. The antibodies included anti-estrogen receptor α (ERα, Santa Cruz Biotechnology, USA), anti-progesterone receptor A (PRA, Proteintech, China), anti-hypoxia inducible factor 1α (HIF1α, R&D, USA), anti-VEGFA (Proteintech, China), anti-angiopoietin 2 (ANGPT2, Abcam, USA), anti-cyclooxygenase 2 (COX2, CST, USA), anti-prostaglandin E receptor 2 (EP2 receptor, Abcam, USA), anti-matrix metallopeptidase 2 (MMP2, Abcam, USA), anti-MMP9 (Abclone, China), anti-tissue inhibitor of metalloproteinase 2 (TIMP2, R&D, USA), anti-FGF2 (Santa Cruz Biotechnology, USA), and anti-β-actin (Proteintech, China). Next, the PVDF membranes were incubated with the fluorescent secondary antibodies (CST, USA) at room temperature for 1 h. Lastly, the protein bands were scanned using the Odyssey Infrared Imaging System (Li-Cor Biosciences, USA).

Isolated uteri were fixed in paraformaldehyde and embedded in paraffin. After dewaxing with xylene and subjecting it to a gradient concentration of alcohol, the antigen was retrieved. The tissue sections were blocked with goat serum for 20 min at room temperature. Then, the sections were incubated with primary antibodies (anti-CD31, Abcam, USA; anti-HIF1α, R&D, USA; anti-COX2, CST, USA; anti-EP2 receptor, Abcam, USA; anti-MMP2, Abcam, USA; anti-MMP9, Abclone, China) for 1 h at room temperature, followed by incubation with the secondary antibody for 1 h at room temperature. After washing, the sections were developed using 3,3′-diaminobenzidine and stained using hematoxylin for about 3 min. Lastly, the slices were processed by dehydration, hyalinization, and sealing. Images were scanned using a NanoZoomer slide scanner (Hamamastu, Japan) and observed using NDP view2 system.