Visualizing the proliferation marker bromodeoxyuridine (BrdU) requires pretreatment of tissue, typically with dilute hydrochloric acid (HCl). We report here that pretreatment by steam heating of paraformaldehyde-fixed tissue sections covered with citrate buffer yields much brighter labeling of BrdU than HCl pretreatment, allows labeling with many antibodies greatly superior to HCl pretreatment, and allows concurrent high-quality labeling of nuclei with the DNA-binding dyes Hoechst, DAPI (4′,6-diamidino-2-phenylindole), and Syto24. Standard use of antigen retrieval by steamer can facilitate new insights into mechanisms regulating normal progenitor and tumor cell proliferation and novel understandings of protein expression through increased sensitivity of immunohistochemical analysis.

Mitotically active progenitor cells from the anterior portion of the forebrain subventricular zone (SVZa), which give rise throughout life to olfactory bulb interneurons, bear processes and express neuronal markers. To understand how rodent SVZa neuronal progenitors coordinate division and process formation, we used time-lapse videomicroscopy to analyse the proliferative behavior of SVZa progenitors in dissociated cell culture continuously for up to five generations. The cell cycle time of these cultured SVZa cells assessed videomicroscopically (cytokinesis to cytokinesis) was similar to the cell cycle time along the rostral migratory stream in vivo (14-17 h). The relationship between process extension, process retraction and cytokinesis was assessed quantitatively for 120 cells undergoing cytokinesis. Although all of these cells had elaborated processes, virtually all of them completely withdrew their processes prior to cytokinesis. Process withdrawal was rapid and tightly coupled to cytokinesis; 50% of the cells studied initiated process retraction within 30 min of cytokinesis and 96% had begun to withdraw their processes within 60 min of cytokinesis. In SVZa progenitor cell lineages, the sequence of process extension, process retraction and division is repeated over multiple generations. This complete withdrawal of processes prior to division differentiates SVZa progenitor cells from the characteristics reported for several other process-bearing types of neural progenitor cells, including sympathetic neuroblasts, cerebral cortical radial glia, and cerebellar and retinal progenitors. Collectively, our findings indicate that SVZa progenitors employ different cellular mechanisms than other neural progenitors to regulate proliferation and differentiation.