Mouse anti gphn

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.

At 3–5 days post in vitro (dpiv), cells that had adhered to the coverslips were washed several times in PBS to remove media and fixed in 4% para-formaldehyde (PFA) for 5 minutes at RT. Cells on coverslips were then washed several times in PBS, and then incubated in blocking solution containing 2% bovine serum albumin (Amresco), and 5% normal goat serum (Sigma) in PBS, for 1.5 hours. Cells were then incubated in primary antibody (mouse anti-GFP, 1:250, EMD Millipore; rabbit anti-PSD95, 1:1000, Abcam; mouse anti-GPHN, 1:1000, Synaptic Systems; rabbit anti-dsRed 1:250,Clontech), diluted in blocking solution, for 1.5 hours at RT. Cells were washed several times in PBS to remove primary antibody. Cells were subsequently incubated in secondary antibody (Goat anti-mouse IgG Superclonal secondary, Alexa588, 1:400, Thermo Scientific; Goat anti-Rabbit IgG, Cy3 conjugate, 1:400, EMD Millipore) with DAPI (1:100; 4′,6-diamidino-2-phenylindole; Invitrogen) diluted in blocking solution and incubated for 30 minutes at RT. Cells were washed in PBS and post-fixed in 4% PFA for 1 minute. Coverslips were mounted on super-frost slides (VWR) in 80% glycerol for imaging.