Mouse anti glyt2

P2, P16-P21, or 8–10 week-old mice were deeply anesthetized with 0.4 mg/g 2,2,2-tribromoethanol (Avertin), then perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (PB), post-fixed overnight at 4 °C, and cryoprotected in 30% glycerol in 0.1 M PB overnight. 12–40 μm tissue sections were cut on a freezing microtome and processed free-floating. Primary antibodies were diluted in PBS containing 5% goat serum and 0.3% triton-X-100, and incubated with tissue sections for 18 hours at 4 °C in a humidified chamber. Primary antibodies included: chicken-anti-GFP (1:500, Aves or Abcam), mouse anti-NeuN (1:200, Millipore), mouse anti-GFAP (1:500, NeuroMab, clone N206A/8 or Millipore, clone GA5), rabbit anti-SLC7A10, N-term (1:250–1:500, Acris, lot #FGI263), rabbit anti-beta-galactosidase (1:500, MP/Cappel), mouse anti-GLYT2 (1:500, Millipore), mouse anti-OLIG2 (1:500, Millipore), mouse anti-PSD95 (1:2000, NeuroMab), and mouse anti-GPHN (1:200, Synaptic Systems). Secondary detection was conducted at room temperature for 2 hours, using the following antibodies: goat anti-rabbit Alexa Fluor 488, 568, or 647, goat anti-mouse Alexa Fluor 488 or 594, goat anti-chicken Alexa Fluor 488, goat anti-guinea pig Alexa Fluor 594 (1:500, all from Invitrogen). Images were acquired using Zeiss Meta 510, Zeiss Axiovis, or Zeiss 800 and 880 Airyscan microscopes.

Spinal cord homogenates were prepared in T-PER tissue protein extraction reagent (ThermoFisher Scientific) containing protease inhibitors (Roche). Protein concentration was measured by BCA assay (ThermoFisher Scientific). Protein equivalents from each sample were boiled in LDS (ThermoFisher Scientific) containing 2.5% β-mercaptoethanol and electrophoresed on 4–12% Bis-Tris acetate gels (BioRad). Protein was transferred to nitrocellulose (BioRad) in transfer buffer containing 25 mM Tris, pH 8.3, 192 mM glycine, 0.1% (w/v) SDS, and 20% methanol. Membranes were blocked for 1 h in 20 mM Tris, 500 mM NaCl, pH 7.5 (TBS) containing 5% milk, then incubated overnight at 4 °C with primary antibodies diluted in TBS-0.1% tween-20 (TBST) containing 5% milk. After washing in TBST, membranes were incubated with HRP-conjugated goat-anti-rabbit or goat-anti-mouse antibodies (GE Life Sciences) and detected using ECL reagents (GE Life Sciences). Primary antibodies included rabbit anti-beta-galactosidase (1:500, MP/Cappel), mouse anti-beta-tubulin (1:1000, Sigma), rabbit anti-SLC7A10, N-term (1:1000, Acris, lot #FGI263), mouse anti-GLYR (1:1000, Synaptic Systems), rabbit anti-VIAAT (1:1000, Aviva), rabbit anti-GLYT1 (1:1000, Aviva), and mouse anti-GLYT2 (1:1000, Millipore). Densitometry was performed using ImageJ.