Anti mmp 9

RIPA lysis buffer (Beyotime) was used to extract total protein. A BCA Protein Quantitative Kit (Beyotime) was adopted to detect protein concentrations. Protein was separated by SDS-PAGE (10% gel) and transferred to PVDF membranes (Millipore, Billerica, MA, USA). For lectin blotting, the membranes were blocked with a carbo-free blocking solution (Vector Labs) and subsequently incubated with biotin-labeled LCA. For western blotting, the membranes were blocked with 5% non-fat milk. Primary antibodies (Absin, Shanghai, China) were used including anti-MMP2, anti-MMP-9, anti-p53, anti-Bcl-xL, anti-CylinD1, anti-BAX and anti-pAkt. The signal was evaluated using an enhanced chemiluminescence detection kit (Beyotime). An antibody against GAPDH (Absin) served as an endogenous reference.

Formalin-fixed paraffin-embedded (FFPF) mouse ectopic endometrial tissues were sectioned into 4 μM thick tissue slices. Slices were deparaffinized in xylene, rehydrated through graded ethanol, and boiled for 10 min in citrate buffer (10 mm, pH 6.0) for antigen retrieval. Endogenous peroxidase activity was suppressed by exposure to 3% hydrogen peroxide for 10 min. Tissue slides were then blocked with 5% BSA (bovine serum albumin; Boster Bioengineering, Wuhan, China), and incubated overnight at 4°C with the following primary antibodies:anti-MMP9 (Absin, China) and anti-VEGFR2 (CST, United States), followed by incubation with corresponding secondary antibody for 20 min. Slides were visualized, adding DAB (3,3′-diaminobenzidine) substrate, counterstained with hematoxylin, and mounted for observation under the microscope.