Cell Proliferation Without Neurogenesis in Adult Primate Neocortex D. R. Kornack and P. Rakic

Supplementary information on the methods:

For immunoperoxidase staining, animals were anaesthetized and perfused with 70% ethanol, and brains were blocked and postfixed overnight at 4°C. Blocks were dehydrated in graded alcohol solutions, cleared in xylene, embedded in paraffin, and serially sectioned at either 8 or 10 m in the coronal plane. Sections were mounted on glass slides and processed for BrdU immunoperoxidase as described previously [D.R. Kornack, P. Rakic, Proc. Natl. Acad. Sci. USA 95, 1241 (1998)]. Briefly, rehydrated sections were placed in 2 N HCl for 1 hr, rinsed, and incubated with a mouse anti-BrdU antibody (Becton Dickinson, San Jose, CA; 1:100) for 30 min at room temperature. The antibody was visualized using a biotinylated horse anti-mouse IgG (Vector Laboratories, Burlingame, CA; 1:200), the Vector ABC Elite kit, and H₂O₂/diaminobenzidine (DAB) with 0.02% cobalt chloride and 0.02% nickel ammonium sulfate to yield a black reaction product. Sections were counterstained using 0.1% basic fuchsin.

For immunofluorescence triple-labeling for BrdU and cell-type markers, we perfused with 0.9% saline, followed by 4% paraformaldehyde (PFA) in 0.1 M PB, pH 7.4. Blocks of brain tissue were postfixed in PFA-PB for 6 hrs at 4 °C, then sunk in graded sucrose solutions to 30%. Blocks were frozen, and coronal cryostat sections (30 or 40-m) were placed in PBS and immediately processed as previously described [D.R. Kornack, P. Rakic, Proc. Natl. Acad. Sci. USA 98, 4752 (2001)]. To detect BrdU, the free-floating sections were pretreated to denature DNA by the following steps: 2 hr incubation in 50% formamide/2 X SSC at 65 °C; 5 min rinse in 2 X SSC; 30 min incubation in 2 N HCl at 37 °C, and 10 min rinse in 0.1 M boric acid, pH 8.5. Sections were then blocked and incubated for 48 hrs at 4 °C with a pooled solution of rat anti-BrdU antibody (Accurate Scientific, Westbury, NY; 1:100), rabbit anti-glial fibrillary acidic protein (GFAP; DAKO, Carpinteria, CA; 1:1000) and either mouse anti-NeuN antibody (Chemicon International, Temecula, CA; 1:125) or mouse anti-class III -tubulin antibody (TuJ1; a kind gift from Dr. A. Frankfurter, University of Virginia; 1:400). After rinsing in blocking buffer, sections were incubated for 2 hrs in a pooled solution of Alexa 488-conjugated goat anti-rat IgG, Alexa 568-conjugated goat anti-mouse IgG (both from Molecular Probes, Eugene, OR; 1:500) and Cy5-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA; 1:200). Sections were mounted and coverslipped with mounting medium. Omitting primary antibodies from the immunohistochemistry processing steps eliminated fluorescence or peroxidase labeling of cells.

Brightfield images were obtained using a CCD camera mounted on a Zeiss microscope. Fluorescent signals were imaged using a confocal laser scanning microscope (Zeiss LSM 510; Carl Zeiss, Thornwood, NY). For obtaining confocal micrographs, each fluorochrome dye within the same field was scanned separately using a quasi-simultaneous mode; this eliminates the possibility of signal "bleeding", which could generate false positive results. For imaging, the emission signals of Alexa-488, Alexa-568 and Cy5 were assigned to the green, red, and blue channels, respectively.