Mouse anti mt1 mmp

Brain tissues were homogenized in RIPA lysis buffer and cleared by centrifugation at 12,000 rpm for 30 min at 4°C. Proteins were separated using SDS-PAGE and transferred onto PVDF membranes. Following blocking, membranes were incubated with either mouse anti-MMP-2 (1:400, Santa Cruz, USA), goat anti-MMP-9 (1:400, Santa Cruz, USA), mouse anti-MT1-MMP (1:400, Santa Cruz, USA), or mouse anti-actin (1:5,000, Abcam, Cambridge, UK) antibodies overnight at 4°C. The next day, membranes were incubated with horseradish peroxidase-conjugated anti-mouse IgG or anti-goat IgG (1:5,000, Promega, USA) secondary antibodies for 2 h at room temperature, and the positive signal was detected with an enhanced chemiluminescent substrate (Thermo Fisher) on a Bio-Rad ChemiDoc XRS digital documentation system. The relative protein expression was compared among rats of different experimental groups.

Endogenous peroxidase was quenched using 3% hydrogen peroxide and nonspecific binding was blocked using 2% bovine serum albumin (BSA) for 1 h. Tissue sections were incubated overnight at 4°C with either goat anti-MMP-9 (1:200, Santa Cruz, USA), mouse anti-MMP-2 (1:200, Santa Cruz, USA), or mouse anti-MT1-MMP (1:400, Santa Cruz, USA) antibodies. To detect cerebral microvascular EC or neural proliferation, sections were incubated with mouse anti-PCNA (1:1400, Cell Signaling Technology) or rabbit anti-vWF (von Willebrand factor; 1:400, Dako, Denmark) for 1 h at 37°C. Subsequently, sections were incubated with biotinylated anti-mouse IgG (1:800, Santa Cruz) or anti-goat IgG (1:800, Santa Cruz) secondary antibodies for 1 h at 37°C followed by the avidin-biotin-peroxidase complex (1:100, Vector Laboratories, USA). Immunoreactivity was detected with diaminobenzidine (Boster Biotech Co., P.R. China).