Biochemical and metabolic analysis of ischemic cerebral tissue is central in stroke investigation and is usually performed in animal stroke models, such as the permanent occlusion of the middle cerebral artery (MCAO) in the rat that we have used. To be sure that the sample is from infarct tissue, it is differentiated from the surrounding normal tissue by staining, usually with 2,3,5-triphenyltetrazolium chloride (TTC), but staining can hamper biochemical colorimetric analysis. We performed this study to avoid this obstacle. A cerebral infarct was provoked in a sample of 10 rats and the brain was cut in coronal sections that were stained with TTC so that the unstained, infarct areas could be delineated in a template of each section in which areas with infarct in all animals were delineated. We calculated infarct coordinates and depth so that the infarct tissue can be sampled without staining. For more precision, the ischemic cortex can be delimited staining its surface before sectioning and cortical tissue into which TTC diffuses can be afterwards discarded, as we had previously measured the TTC diffusion depth in rat brains.