A method for identifying viable and damaged neurons in adult mouse brain slices

The cell survival assay is a commonly used technique for studying cellular mechanisms and degree of neuroprotection following cerebral ischemia. The in vitro preparations for studying ischemia are often hypoxic models induced by oxygen and glucose deprivation (OGD). In vitro studies have been carried out using embryonic/neonatal neuronal cell cultures to estimate the ratio of viable to damaged neurons and the degree of neuroprotection following OGD. Brain slices are more physiologically relevant preparations compared to cell cultures. However, no simple assay is currently available to identify both damaged and viable cells in the same brain slice. In addition, since stroke-related ischemic neuronal injury occurs primarily in adults, adult brain slices exposed to OGD may be beneficial for studying cerebral ischemia. Here, we describe a reliable double-labelling procedure using propidium iodide (PI) and anti-neuronal nuclei (NeuN) antibody to detect both damaged and viable neurons in the same adult mouse brain slice subjected to OGD. In addition to the cerebral ischemia, this method may prove useful in other neuronal stress models.