Ultrastructural identification of dividing cells in the adult mammalian central nervous system

In most parts of the adult mammalian central nervous system cell division is a relatively rare event, which makes it difficult to study at the ultrastructural level. We designed a protocol for reliable ultrastructural identification of proliferating cells in a tissue volume using DNA-incorporated 5-bromo-2-deoxyuridine (BrdU) as a marker. After BrdU administration the tissue is fixed and embedded in hydrophilic resin (LR Gold) and then cut in serial 1–2 μm sections and mounted on glass slides. BrdU is detected at the light microscopic level using immunogold labeling followed by silver enhancement, according to a standard procedure. After detection of labeled nuclei the section is reembedded in resin on the same glass slide. The glass is then dissolved in hydrofluoric acid and labeled cells cut in ultrathin sections for further ultrastructural analysis. The technique was tested and refined in sections of the intestine containing numerous dividing cells and, once optimized, was then applied to identify the ultrastructure of slowly proliferating putative stem cells in the adult mouse spinal cord.