S2532

Mice were daily given an IP injection of 5-bromo-2′-deoxyuridine (BrdU; 50 mg/kg, Sigma-Aldrich, St. Louis, MO, USA) for 12 days, beginning after stereotaxic surgery [14 (link)]. R6/2 mice euthanasia performed at 6 weeks (early-HD stage) and 13 weeks of age (late-HD stage) by transcardial perfusion with cold 1X PBS, followed by 4% paraformaldehyde (PFA). Harvested brain tissues were cryosectioned 16-μm thick slices, and immunohistochemical staining was performed on four sections, representing a range of more than 128 μm. The tissue sections were stained with the following antibodies: cell proliferation marker; BrdU (1:200, abcam, ab6326) and Ki67 (1:400, Leica Biosystems, NCL-Ki67p); OCT4 tagging marker HA (1:400, CSF, 3724S); OCT4 (1:100, santacruz, sc-5279); neuron-specific class III β-tubulin (βIII-tubulin, 1:400, abcam, ab18207) and mature neuronal marker NeuN (1:400, Millipore, MAB377); glial fibrillary acidic protein (GFAP, 1:400, abcam, ab10062) and s100β (1:400, Sigma, S2532); Nestin (1:400, abcam, ab6142); neural/glial antigen 2 (NG2, 1:200, Millipore, ab5320) and dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP-32, 1:400, cell signaling technology, 2306). The stained sections were observed by confocal microscopy (LSM700, Zeiss, Gottingen, Germany) and analyzed using ZEN black and blue edition (Zeiss, Gottingen, Germany).