At 24 h after MCA occlusion and after neurological assessment, the mice were deeply anesthetized with isoflurane, and brains were harvested rapidly after perfusion with cold phosphate buffered saline and cold 4% paraformaldehyde. For histological analysis, brains were cut into 40 micron coronal sections with a vibratome. Coronal sections taken at rostral 2.34 mm, 1.34 mm, 0.26 mm, and caudal −0.70 mm, −1.70 mm, and −2.70 mm to bregma were assessed by cresyl violet staining. The infarct area was measured by a blinded observer using digital imaging and image analysis software (Image J 1.37v, Wayne Rasband, available through National Institutes of Health). Infarct area was corrected for edema using the method of Swanson et al.24 The person assessing infarct areas was blinded to the genotype of each brain. Cresyl violet staining allows the same brains to be used for additional histological analysis.
For ICI 118,551-treated mice and vehicle controls brains were removed, sectioned, and incubated in 2% 2,3,5-triphenyltetrazolium chloride (TTC) in saline for 20 min at 37°C. To determine infarct volume by TTC staining, six slices per rat were analyzed by a blinded observer using the National Institutes of Health Image program as previously described.24,25
For ICI 118,551-treated mice and vehicle controls brains were removed, sectioned, and incubated in 2% 2,3,5-triphenyltetrazolium chloride (TTC) in saline for 20 min at 37°C. To determine infarct volume by TTC staining, six slices per rat were analyzed by a blinded observer using the National Institutes of Health Image program as previously described.24,25
