Rabbit anti mmp2

Total proteins were extracted by using radioimmunoprecipitation assay buffer reagent (RIPA, Beyotime, China) and phenylmethanesulfonyl fluoride (PMSF, Sigma, USA). A Bicinchoninic acid (BCA) protein assay kit (Beyotime, China) was used to measure the protein concentrations. Proteins from each sample were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto the Polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The lysates of tissues or cells with equal weight were separated as aforementioned. After blocking in the 5% skim milk with tris buffered saline tween (TBST, Boster, China), the transferred membranes were incubated in the primary antibodies at 4°C overnight and then washed by TBST (TBS+Tween) for three times for 15 minutes each. Subsequently, the transferred membranes were incubated with secondary antibodies at RT for 1h. After extensive washing in TBST, immunoblotting was visualized using the enhanced chemiluminescence (ECL) development kit (Thermo Scientific, USA) and analyzed with Quantity-one software (Bio-Rad, USA). Antibodies used: rabbit anti-MMP-2, rabbit anti-MMP-9, rabbit anti-Bax and rabbit anti-Bcl-2 were purchased from Abcam UK.

Slides with the frozen aorta sections were air-dried and fixed in 4% paraformaldehyde for 15 minutes, washed 3 times (5 min each time) with PBS, and then blocked with 5% normal goat serum for 60 min at room temperature in a humid chamber. Sections were subsequently incubated overnight at 4°C with the following primary antibodies applied: mouse anti-α-SMA (1 : 400), mouse anti-CD68 (1 : 500; Abcam, USA), mouse anti-MCP1 (1 : 500; Santa Cruz Biotechnology, USA), rabbit anti-MMP2 (1 : 500; Abcam, USA), rabbit anti-MMP9 (1 : 500; Abcam, USA), rabbit anti-IL6 antibody (1 : 300; Abcam, USA), and rabbit anti-TNFα (1 : 500; Abcam, USA). Sections were washed 3 times (5 min each time) with PBS, Alexa Fluor 488 goat anti-mouse IgG (1 : 500; Invitrogen, USA), and Alexa Fluor 594 goat anti-rabbit IgG (1 : 500; Invitrogen, USA), and the sections were incubated in secondary antibodies for 60 minutes at room temperature in a light-protected humid chamber. The sections were washed 3 times (5 min each time) with PBS and stained with 4,6-diamidino-2-phenylindole (DAPI, 1 : 30, Sigma, USA). Slides incubated with secondary antibodies were used as negative controls.

Protein preparation and Western blot were performed as described previously [20 (link)]. The antibodies for Western blot analysis were as follows: rabbit anti-RAI2 (Cell Signaling Technology, Danvers, MA,USA), rabbit anti-MMP2 (Abcam, Cambridge, UK), rabbit anti-MMP7 (Abcam, Cambridge, UK), rabbit anti-MMP9 (Abcam, Cambridge, UK), rabbit anti-caspase 3 (Abcam, Cambridge, UK), rabbit anti-cleaved caspase 3 (Abcam, Cambridge, UK), rabbit anti-AKT (Bioworld Technology, Beijing, China), mouse anti-phospho-AKT^ser473 (Cell Signaling Technology, Danvers, MA,USA), anti-E-cadherin (BD Biosciences, Franklin Lakes, NJ, USA), and anti-vimentin (Cell Signaling Technology, Danvers, MA,USA). Rabbit anti-actin (Cell Signaling Technology, Danvers, MA, USA) was used as a control. Each experiment was repeated for three times.

Brain coronal frozen sections (20 μm) were produced in the same manner as described above. The sections were blocked with 10% normal goat serum (Vector Laboratories, Burlingame, CA, USA) at room temperature for 1 h and then incubated with primary antibodies overnight at 4 °C. The primary antibodies used were as follows: rabbit anti-occludin (1:50, Proteintech Group Inc., Rosemont, USA), rabbit anti-ZO-1 (1:50, Proteintech Group Inc.), rabbit anti-claudin-5 (1:200, Affinity Biosciences, OH, USA), and mouse anti-vWF to label endothelial cells (1:50, Millipore, Temecula, CA, USA); or rabbit anti-MMP-2 (1:100, Abcam Inc., Cambridge, MA, USA), rabbit anti-MMP-9 (1:1000, Cell Signal Technology, Boston, USA), and mouse anti-GFAP to label astrocyte cells (1:100, Cell Signal Technology). Brain sections were rinsed with PBS and then incubated with Cy® 3 conjugated goat anti-rabbit IgG antibody (1:500, Invitrogen, Carlsbad, CA, USA) and Alexa Fluor 488 conjugated goat anti-mouse IgG antibody (1:300, Molecular Probes) for 1 h at room temperature. All nuclei were stained with DAPI (molecular probes). After being washed, the sections were observed under a laser scanning confocal microscope (FV10i, Olympus, Tokyo, Japan).