A Painless Technique For Alizarin

2, 0.7, and 0.2 mm rostral to the bregma; Paxinos and Watson, 1982) of each animal, and four areas in the ipsilateral peri-infarcted cortex in each section were chosen randomly and captured at ��100 with a confocal laser microscope. We confirmed the border between the ischemic core and the peri-infarct lesion through cresyl violet staining of adjacent sections according to a previously described method (Omori et al., 2002) and measured the area between astrocyte endfeet and the basement membrane of each blood vessel, as well as the length of each blood vessel. Then, the area-to-length ratio was calculated as the ��vascular Alizarin dissociation index�� (Yamashita et al., 2009). In the same way, the area between astrocyte endfeet and pericyte and the area between pericyte and vascular endothelial cells were measured, and the area-to-length ratio was calculated in each blood vessel. Gelatin zymography was performed using frozen brain tissue from the cerebral cortex. Frozen brain samples were homogenized in 10�� volume lysis buffer (150 mM NaCl, 1% sodium dodecyl sulfate [SDS], 0.1% deoxycholic acid, and 50 mM Tris-HCl, pH 7.4) containing protease inhibitors. After centrifugation at 9,000g for 15 min at 4��C, the supernatant was collected. Total protein concentration of each supernatant was spectrophotometrically selleck screening library determined using the Bradford assay (Ultrospec 3100 Pro; GE Healthcare, Tokyo, Japan). The activity of MMP-9 in each sample was measured using a gelatin-zymography kit (Primary Cell, Sapporo, Japan) according to the manufacturer's instructions. In brief, each sample containing 20 ��g protein was diluted with the homogenizing buffer in the kit, mixed with an equal volume of sample buffer, and loaded for electrophoresis for 2 hr. The gels were washed and incubated for 24 hr in incubation buffer at 37��C, then stained with Coomassie blue and scanned. Quantitative densitometric analysis was performed in Image J software. All data are presented as mean?��?SD. Statistical analyses were performed with one-factor analysis of variance followed by Tukey-Kramer's postcomparison test. Differences with a probability value of P?Selleck SB203580 significant. Mean body weight and systolic and diastolic blood pressure were not significantly different among the three groups (Table 1). The paraparesis score was significantly improved in the D?+?tPA group (3.7?��?2.3; *P?