Mouse anti gad67

Immunohistochemistry on 12 μm thick cryostat sections of lumbar level (L) 4 to 5 spinal cord was performed as previously described (Betley et al., 2009 (link)). Rabbit anti-βgal (gift from J. Sanes) (Gray and Sanes, 1991 (link)), rabbit anti-ChAT (generously provided by S.B.-M. and T.M.J., unpublished data), rabbit anti-GAD65 1:50,000 (Betley et al., 2009 (link)), mouse anti-GAD67 1:10,000 (Millipore), rabbit anti-GAD67 1:10,000 (Betley et al., 2009 (link)), chicken anti-GFP 1:1,000 (Millipore), sheep anti-GFP 1:1,000 (Molecular Probes), rat anti-NB2 (1A6) 1:4 (Shimoda et al., 2012 (link)), chicken anti-Pv 1:10,000 (generously provided by S.B.-M. and T.M.J., unpublished data), rabbit anti-RFP (Rockland), rabbit anti-Shank1a 1:64,000 (Betley et al., 2009 (link)), rabbit anti-Shank1a 1:1,000 (Millipore), mouse anti-Syt1 1:100 (ASV48, Developmental Studies Hybridoma Bank), and guinea pig anti-vGluT1 1:32,000 (Betley et al., 2009 (link)).

Antibodies used in these studies were as follows: rabbit anti-calbindin (diluted 1:2500 for immunohistochemistry (IHC); Swant), rabbit anti-calretinin (diluted 1:1000 for IHC; Millipore), rabbit anti-GFP (diluted 1:500; Life Technologies), mouse anti-GAD67 (diluted 1:1000 for IHC; Millipore), rabbit anti-Iba1 (diluted 1:500 for IHC, WAKO), rabbit anti-Vasoactive Intestinal Peptide (VIP; diluted 1:150 for IHC; Immunostar). All fluorescent secondary antibodies for IHC were from Life Technologies (diluted 1:1000).

Rats were anesthetized with 5% chloral hydrate anesthesia (7.5 ml/kg, i.p.) and transcardially perfused with saline and 4% PFA. Brains were post-fixed in 4% PFA overnight and coronal sections were serially cut at 40 μm on a Leica VT1200S vibratome. Immunohistological staining was performed on free-floating sections. Slices were blocked in a solution containing 0.4% Triton X-100 and 10% donkey serum and then stained with rabbit anti-GFP (1:500, Invitrogen), mouse anti-CamkIIα (1:500, Thermo, Rockford, lL, USA) and mouse anti-GAD67 (1:2000, Millipore) antibodies before incubation with the appropriate fluorescence-conjugated secondary antibodies.

Human NSCs and differentiated neuronal cells were seeded on chamber slides. The cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.5% Triton X-100 in TBS for 5 min at room temperature. TBS with 2% FBS was used for blocking. The cells were incubated with the following primary antibodies at 4 °C overnight: rabbit anti-SOX2 (1:200, Cell Signaling, CA, USA), mouse anti-nestin (1:200, Millipore, MA, USA), rabbit anti-MAP2 (1:200, Millipore, MA, USA), mouse anti-Tuj1 (anti-neuron-specific class III β-tubulin, 1:200, Millipore, MA, USA), mouse anti-GAD67 (1:200, Millipore, MA, USA), rabbit anti-active caspase 3 (1:200, Millipore, MA, USA), rabbit anti-LC3A/B (1:200, Cell Signaling, CA, USA), mouse anti-EV-A71 3D (1:500, Genetex, CA, USA) or rabbit anti-EV-A71 3A (1:500). The cells were then washed with TBS and incubated with the following secondary antibodies for 1 h at room temperature: DyLight 488-conjugated goat anti-rabbit secondary antibody or DyLight 594-conjugated donkey anti-mouse secondary antibody (1:1,000, Jackson ImmunoResearch Laboratories, Pennsylvania, USA). The cells were then washed with TBS, and cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, MO, USA). Images were collected with an Olympus BX51 fluorescence microscope (Olympus, Tokyo, Japan) and an LSM 510 microscope (Zeiss, Jena, Germany).