Mab4224

CS sections were washed with PBS to remove residual OCT/sucrose and then blocked in a solution consisting of PBS with 10% normal donkey serum (NDS), 0.3% Triton X-100 and 1% BSA for 1 hour at room temperature. The sections were then incubated with primary antibodies diluted in a solution consisting of PBS with 2% NDS and 0.1% Triton X-100 overnight at 4°C. The following primary antibodies were used for staining: anti-PAX6 (mouse, 1:300, BioLegend, PRB-278P), anti-CTIP2 (rat, 1:300, abcam, ab18465), anti-TBR2 (mouse, 1:100, R&D Systems, MAB6166), anti-SOX9 (goat, 1:300, R&D Systems, AF3075), anti-SSTR2 (mouse, 1:100, R&D systems, MAB4224), anti-MAP2 (guinea pig, 1:200, Synaptic Systems, 188 004), and anti-TUBB3 (rabbit, 1:200, Cell Signaling TECHNOLOGY, #5568S). Sections were washed with PBS to remove primary antibodies, and incubated with secondary antibodies (Alexa Fluor dyes, Life Technologies) diluted 1:1000 in PBS with 2% NDS and 0.1% Triton X-100 for 1 hour at room temperature. Sections were washed with PBS and nuclei were stained with Hoechst 33258 (Thermo Fisher Scientific, H3569) for 5 minutes at room temperature. Sections were mounted on glass slides with Aqua-Poly/Mount (Polysciences, Inc., 18605-5) and imaged with a Leica TCS SP8 confocal microscope. Images were processed with ImageJ (Fiji).

hCS and cCS were dissociated and plated down for calcium imaging and immunocytochemistry experiments as previously described^{15 (link)}. Briefly, 15–20 CS per cell line were incubated in 30 U/mL Papain (Worthington, LS 03126), 1 mM L-cystine (Sigma, C7880) and 125 U/ml DNase I (Worthington, LS002007) for 45 minutes. Following a series of Papain-inactivating washes, CS were single-cell triturated and plated down in neural media supplemented with BDNF (20 ng/mL) and NT-3 (20 ng/mL) on glass coverslips coated with poly-l-ornithine and laminin (Sigma-Aldrich) at a density of 90,000-100,000 cells per coverslip. Prior to plating, cells for these imaging experiments were first immunopanned for THY1 and HEPACAM for use in other experiments. Calcium imaging and immunocytochemistry experiments were performed within 9 and 30 days of dissociation. Coverslips were fixed for immunostaining as follows: a solution of warmed 4% PFA/4% sucrose/PBS was added to the wells with coverslips to be fixed for 20 minutes. Cells were then washed with PBS and stored in PBS at 4° C. Staining was performed as described above, and the following primary antibodies were used for staining: anti-SSTR2 (mouse, 1:100, R&D systems, MAB4224), anti-MAP2 (guinea pig, 1:200, Synaptic Systems, 188 004).