?(Fig.55,indicate m-Msi-1-positive and NG2-negative cells. development and maintenance of neural cells. During CNS development, neurons and glial cells are thought to be generated from common neural precursor cells (multipotent CNS stem cells) (Davis and Temple, 1994). Our previous study has suggested that m-Msi-1 is expressed predominantly Gatifloxacin mesylate in proliferating multipotent neural precursor cells that may give rise to neurons and/or glia, but is not expressed in newly generated postmitotic neurons (Sakakibara et al., 1996). The expression of m-Msi-1 is largely downregulated with the successive progression of neurogenesis. However, a considerable amount of m-Msi-1 transcripts is present in the brain during postnatal development, and low level expression persists into adulthood (Sakakibara et al., 1996). Recent studies have indicated the presence of neural precursor cells in postnatal neural development. To further study the role of m-Msi-1 in neural precursor cells in the postnatal and adult CNS, the spatiotemporal distribution of m-Msi-1-expressing cells was examined. The m-Msi-1 protein can be detected in proliferating neuronal and/or glial precursor cells in the SVZ and in cells of the astrocyte lineage in the postnatal and adult CNS. These observations suggest also that m-Msi-1 may be an excellent marker for protoplasmic astrocytes. MATERIALS AND METHODS ICR mice and Wistar rats used throughout the experiments were purchased from Charles River Japan Inc. The day of birth was designated as postnatal day (P) 0. Pups and adults (anesthetized by ether inhalation) were perfused through the left ventricle with 4% paraformaldehyde in 0.1m PBS, pH 7.4, and then the brains were dissected and post-fixed overnight at 4C in the same fixative. For paraffin sectioning, tissue blocks were rinsed twice with PBS for 30 min each time and dehydrated through a series of increasing concentrations of ethanol. After dehydration, they were cleared with chloroform and xylene and then embedded in paraffin (Tissue Prep, Fisher, Pittsburgh, PA) at 62C. Sections (4 m) were cut on a microtome and mounted on gelatin/chrome-coated slides. Paraffin sections were used for hematoxylinCeosin and immunohistochemical staining with a single primary antibody. For cryosectioning, sample blocks rinsed with PBS were cryoprotected in 30% sucrose in PBS overnight at 4C, embedded in O.C.T. compound (Tissue Tek, Miles, Elkhart, IN), and frozen on dry ice. Cryostat sections (12 m) were cut and affixed to glass slides precoated with gelatin/chrome. These sections were used for double-immunostaining. The following primary antibodies were used: m-Msi-1 (affinity-purified rabbit polyclonal antibody, used at 1:500 dilution) (Sakakibara et al., 1996); Hu proteins (mouse monoclonal IgG2B, clone 16A11, which binds to Hu proteins including HuD, HuC, and HelN-1, supplied by Dr. M. F. Gatifloxacin mesylate Marusich, University of Oregon; used at 1:500 dilution) (Marusich et al., 1994); nestin (mouse monoclonal IgG, clone RE6C96 supplied by Dr. M. Ogawa, Kochi Medical School, Japan); ascitic fluid used at 1:1000 dilution (Miyata and Ogawa, 1994); proliferating cell nuclear antigen (PCNA) (mouse monoclonal IgG1, used at 1:200 dilution; Novocastra Laboratories); glial fibrillary Gatifloxacin mesylate acidic protein (GFAP) (mouse monoclonal IgG1, clone G-A-5, used at 1:400 dilution) (Sigma, St. Louis, MO); 2, 3-cyclic nucleotide-3-phosphohydrolase (CNPase) (mouse monoclonal IgG1, clone 11C5B, used at 1:100 dilution) (Sigma); bromodeoxyuridine (BrdU) (mouse monoclonal IgG1, clone BU-33, used at 1:5000 dilution) (Sigma); MacI (mouse monoclonal IgG1, used at 1:100 dilution) (Boehringer Mannheim, Indianapolis, IN), platelet-derived growth factor -receptor (PDGF-R) (rat monoclonal IgG, a kind gift from Dr. S.-I. Nishikawa, Kyoto University, Japan; used at 1:5000 DP2 dilution) (Takakura et al., 1996), and NG2 (mouse monoclonal IgG1, a kind gift from Dr. W. D. Richardson, University College London, UK; used at 1:100 dilution) (Stallcup and Beasley, 1987). Gatifloxacin mesylate Secondary and tertiary reagents were obtained commercially from Vector Laboratories (Burlingame, CA) or Jackson ImmunoResearch (West Grove, PA). The specificity of the polyclonal antibody to m-Msi-1 was confirmed by the immunoblot analysis of the knock-out mice (our unpublished results). Immunolabeling with a single primary antibody was performed with an avidinCbiotinCperoxidase technique..