Anti smemb

Immunofluorescence staining was performed on the fixed frozen brain sections as previously described^{26 (link)}. Sections were incubated overnight at 4°C with the anti-SMemb (Abcam), or the anti- alpha-SMA (Santa Cruz Biotechnology) with the anti-vWF (Millipore, Temecula, CA), followed by appropriate fluorescence dye-conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA) for 2 hours at room temperature. The sections were visualized with a fluorescence microscope (Olympus OX51, Tokyo, Japan).

At 24 hours following ICH, mice were perfused under deep anesthesia with cold phosphate-buffered saline (PBS, pH 7.4), then infused with 10% formalin. Brains were removed and fixed in formalin at 4°C for a minimum of 3 days. Samples were dehydrated with 30% sucrose in phosphate-buffered saline (PBS, pH 7.4) and the frozen coronal slices (10 μm thick) were sectioned in cryostat (CM3050S; Leica Microsystems). Immunofluorescence was performed as previously described.^{22 (link),36 (link)} Anti-PDGFR-β antibody (1:100, Santa Cruz), anti-p-p38 MAPK (1:100, Santa Cruz), anti-p-MK2 (1:100, Santa Cruz) anti-ICAM-1(1:100, Santa Cruz), anti-SMemb (1:100, abcam) were incubated separately with primary antibodies: anti-smooth muscle actin (1:1000, Santa Cruz) overnight at 4°C. It was then incubated with the appropriate fluorescence conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA). The slices were observed underneath a fluorescence microscope (Olympus BX51, Olympus Optical Co. Ltd, Japan), and pictures were taken with software MagnaFire SP 2.1B (Olympus, Melville, NY).

Western blot tests were performed as reported previously^{26 (link)}. The circle of Willis blood vessels and BAs were harvested under a microscope and homogenized. Primary antibodies used were anti-SMemb (Abcam), the anti- alpha-SMA, the anti-ILK, the anti- phospho-FAK, the anti- total-FAK, the anti-β-actin, the anti-tubulin (Santa Cruz Biotechnology).