This paper presents the determination of an optimal temperature for delayed 2,3,5-triphenyltetrazolium chloride (TTC) staining. Twenty-one rats were subjected to right middle cerebral artery embolic stroke and sacrificed 96 h following ischemia. The brains were harvested and stained immediately after sacrifice or stored for 8 h at 21-23 degrees C or 4 degrees C, respectively. The stained sections were scanned and infarct volume calculated. The quality of staining, distinction of borders between infarcted and non-ischemic tissue and ease of differentiating ischemic tissue in colored and grayscale images was assessed. The present study indicates that results of TTC obtained immediately after animal sacrifice, or delayed TTC staining while storing the brains at room temperature or 4 degrees C are comparable.