Mab4a

For protein separation, 11% PAM (polyacrylamide) gels were used. Gels were run at 150 V for 90 min. Proteins were transferred to nitrocellulose (GE Healthcare, Freiburg, Germany) using a wet blot transfer system (transfer buffer: 25 mM TRIS, 192 mM glycine, 10% ethanol) (Bio-Rad, Hercules, CA, United States). For GlyR protein transfer, 2 h at 200 mA were used. For larger proteins, e.g., cadherin, overnight blotting at 100 mA was performed. Membranes were blocked for 1 h with 5% BSA in TBS-T (TBS with 1% Tween 20). Primary antibodies were incubated overnight at 4°C. Proteins were detected with the pan-α antibody for GlyRs (mAb4a, Synaptic Systems, Göttingen, Germany, Cat. No. 146 011, 1:500) and pan-cadherin (Cell Signaling Technology, Danvers, MA, United States, Cat. No 4068, 1:1500) served as loading control. Signals were detected using the SuperSignal West (Thermo Fisher Scientific, Waltham, MA, United States).

For protein separation, 11% polyacrylamide gels were used. Gels were run at 150 V for 90 min. Proteins were transferred to a nitrocellulose membrane (GE Healthcare, Freiburg, Germany) using a wet blot transfer system (Bio-Rad, Hercules, CA, USA). For GlyR protein transfer, 2 h at 200 mA were used. Cadherin transfer required overnight blotting at 100 mA. Membranes were blocked for 1 h with 5% BSA in TBS-T (TBS with 1% Tween 20). Primary antibodies (pan-α antibody for GlyRs, MAb4a, Synaptic Systems, Göttingen, Germany, 1:500 in TBS-T and pan-cadherin, Cell Signaling Technology, Danvers, MA, USA, 1:1.500 in TBS-T) were incubated overnight at 4°C. Signals were detected using SuperSignal West Pico (Thermo Fisher Scientific, Waltham, MA, USA).

Rats were deeply anesthetized with pentobarbital (6%) and perfused transcardially with phosphate buffer (PB) 0.1 M (pH 7.3) and paraformaldehyde (4%) supplemented with 0.25% glutaraldehyde. The brain was post-fixed overnight in the same solution and finally cryoprotected with 30% sucrose in 0.1M PB. Brains were sliced (30 μm) using a vibratome and immunostainings were performed as previously described [13 (link)]. Immunostained sections were observed with a ZEISS LSM 710 confocal microscope (ZEN software) with 405 diode, 488 and 633 laser lines. All image acquisitions were achieved using 40X and 60X magnification and variable numerical zooms. Channels were acquired with sequential scanning with a pinhole aperture of 1 airy unit each. Images were then analyzed using ImageJ software. Staining specificity was verified with negative controls in which primary or secondary antibodies were omitted. Affinity-purified primary antibodies were: mouse monoclonal anti serine racemase (Transduction Laboratories: 1/1000), rabbit polyclonal anti-d-serine (GemacBio: 1/1000), mouse polyclonal anti-GFAP (Sigma: 1/2000, clone G-A-5), mouse monoclonal anti-glyR (mAb4a, Synaptic Systems 1/100). Secondary antibodies were obtained from Invitrogen: Alexa 488-Goat anti-mouse (1/1000), Alexa 633-Goat anti-rabbit (1/1000) and from Life Technologies: FITC conjugated mouse antibody (1/400).