Anti vwf

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).

Anesthetized pups were transcardially perfused with 0.1 M PBS followed by 4% formaldehyde solution (PFA) 48 hours after HI. The brains were then removed, postfixed (4% PFA, 4°C, 24 hr), and then transferred into a 30% sucrose solution for 2 days. The cryoprotected brains were sectioned at a thickness of 10 µm with a cryostat (LM3050S, Leica Microsystems Inc, IL, USA) for double fluorescence staining. The sections were then washed three times with 0.1 M PBS and were incubated with blocking solution (10% normal goat serum in 0.1 M PBS) for 2 hours at room temperature following 0.1%Triton X-100 (37°C, 30 min). Primary antibodies anti-GFAP (1:200, Millipore, Billerica, MA, USA), anti-vWF (1:200, Millipore, Billerica, MA, USA), anti-G-CSFR (1:100, Santa Cruz Biotechnology, Inc., CA, USA), anti-p-GSK-3β (Tyr216,1:200, abcam, Cambridge, MA,USA), anti-β-Catenin (1:200, abcam, Cambridge, MA, USA), anti-MPO (1:100, Santa Cruz Biotechnology, Inc., CA, USA), and DAPI (Vector Laboratories Inc. Burlingame, CA, USA) were applied (4°C, overnight).
The sections were then washed with 0.1 M PBS and incubated for 2 hours with secondary antibodies (1:200, anti-mouse IgG labeled with Alexa Fluor-488, anti-rabbit IgG labeled with Alexa Fluor-568, Jackson ImmunoResearch Laboratories, Inc. West Grove, PA, USA) at room temperature. The stained slices were observed with an OLYMPUS BX51 microscope.