A mixture of pentobarbital and phenytoin sodium (Euthasol, 180 mg kg−1, Virbac) was given intraperitoneally to deeply anesthetize (EEG flatlined) and kill (heart rate and respiration fell to zero) mice and rats. The mice and rats were then transcardially perfused with saline followed by 4% paraformaldehyde in phosphate buffer using a peristaltic pump. Brain, aorta, kidneys and femoral vessels were then dissected and incubated with 30% sucrose in phosphate buffer. Then the tissues were placed on dry ice and embedded into optimal cutting temperature (OCT) compound (Tissue-Tek). Tissue sections (40 μm thick) were cut on a vibratome. As we had to perform immunohistochemistry on multiple adjacent components of the vessel wall, it was necessary to ensure that paraformaldehyde fixation itself did not influence antibody binding to vessel lamina. Therefore in a few mice and rats, we performed histology without paraformaldehyde perfusion. In these instances, tissues were dissected immediately after killing, placed on dry ice, embedded in OCT compound and stored at −80 °C. OCT-embedded frozen tissue was cryosectioned (8–10-μm-thick sections). Postmortem blocks of macaque monkey cerebral cortex were also cut on a vibratome (5- and 40-μm-thick sections). Human aorta and coronary samples were flash-frozen directly by ILSbio before our study began and before our request for such samples from them. ILSbio obtained these tissues from healthy subjects with a postmortem interval of ~2 h or less. Human tissue samples were cut in 5-μm-thick sections. During our study, no local or national human tissue bank had cerebral cortex tissue sections from healthy subjects with short postmortem intervals. Short postmortem interval samples from healthy human tissue samples appeared to be necessary for robust labeling of artery and arteriole walls by Alexa 633 because our anecdotal observations from postmortem tissue samples that had undocumented postmortem intervals, stored in fixative for years and/or associated with extensive neurodegenerative pathology did not yield bright artery- or arteriole-specific labeling with Alexa 633. Our rat and mouse tissues essentially had zero postmortem intervals to transcardial paraformaldehyde perfusion or OCT embedding and flash-freezing. Furthermore, rodent samples were not stored in paraformaldehyde or other fixative for months. We observed no difference in the quality of Alexa 633 binding to artery and arteriole walls using our two methods of histological preparation of rat or mouse tissue. For flash-frozen human tissue, the quality of Alexa 633 labeling of artery walls was equally good irrespective of whether the dye was applied to unfixed or acetone-fixed tissue sections or whether endogenous peroxidase was quenched.