Rabbit anti gaba

Primary neurons and coronal brain sections were washed in PBS containing 0.3% Triton-X100, blocked in the same solution containing 5% BSA, and then incubated in primary antibodies for 1–2 h. They were then washed three times and then incubated with secondary antibodies containing fluorophores for 1 h before three final washes. Primary antibodies included rabbit anti-GABA 1:500 (Sigma A2052), rabbit anti-parvalbumin 1:400 (Swant, PV-27). Alexa-conjugated secondary antibodies (Thermo Fisher) were used to detect primary antibodies. Native tdTomato fluorescence was imaged, and in vitro primary, MGE cultures were also labeled for DAPI using NucBlue Fixed Cell ReadyProbes (Thermo Fisher, R37609). A Nikon eclipse Ts2R microscope (Photometrics CoolSnap dyno camera) and Leica DM2000 microscope (DFC3000G camera) captured the primary culture and transplant images, respectively.

Immunohistochemistry was done as described⁹¹ (link) except that the blocking step was overnight at 4°C. Primary antibodies: mouse anti-nc82 (DSHB, AB_2314866) 1:40, chicken anti-GFP (Abcam, ab13970) 1:1000, mouse anti-ChAT4B (DSHB, AB_528122), rabbit anti-GABA (Sigma, A2052). Secondary antibodies: Alexa-568 anti-mouse (Invitrogen) 1:400, Alexa-488 anti-chicken (Invitrogen) 1:400, Alexa-633 anti-mouse (Invitrogen) 1:400, Alexa-568 anti-rabbit (Invitrogen) 1:400.
Prolonged incubation (2-3 days at 4°C) with primary and secondary antibodies was required for homogeneous staining. Specimens were whole mounted in Vectashield (Vector Labs) on charged slides to avoid movement. Confocal stacks were acquired using a Zeiss 780 confocal microscope. Brains were imaged at 768 x 768 pixel resolution every 1 μm (0.46 x 0.46 x 1 μm) using an EC Plan-Neofluar 40x/1.30 oil objective and 0.6 zoom factor. All images were acquired at 16-bit color depth. Maximum projections of z stacks were made in Fiji.⁸⁹ (link)

Antibody staining and immunohistochemistry were performed as previously described (Wang et al., 2004 (link)). The following primary antibodies were used: rabbit anti-GFP (Invitrogen,  Carlsbad,  CA, 1:1,000), mouse anti-GFP (Invitrogen, Carlsbad,  CA,  1:1,000), mouse anti-nc82 (Hybridoma Bank,  Iowa  City, IA, 1:500), rabbit anti-RFP (Clontech, Mountain  View,  CA,  1:500); rabbit anti-GABA (Sigma-Aldrich,  St.  Louis,  MO, 1:1,000); rabbit anti-FruM (1:100). For GRASP experiments, we used a mouse monoclonal antibody that specifically recognizes reconstituted GFP (Sigma-Aldrich,  St.  Louis,  MO, 1:200). Secondary antibodies were Alexa Fluor goat anti-mouse 488, goat anti-rabbit 488, goat anti-mouse 568, goat anti-rabbit 568 (Life Technologies, Carlsbad,  CA,  1:100).

Reprogrammed neurons were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) for 20 min at room temperature (RT), then permeabilized with 0.2% Triton X-100 for 10 min at room temperature. Cells were blocked with 1% goat serum, incubated with primary antibodies at 4°C overnight, then incubated with secondary antibodies for 1 hr at RT. Primary antibodies used for immunocytochemistry included chicken anti-MAP2 (Abcam Cat# ab5392 RRID:AB_21381531; 1:10,000 dilution), mouse anti-β-III tubulin (Covance Research Products Inc Cat# MMS-435P RRID:AB_2313773; 1:5000), rabbit anti-β-III tubulin (Covance Research Products Inc Cat# PRB-435P-100 RRID:AB_291637; 1:2000), chicken anti-NeuN (Aves Labs Cat# NUN RRID:AB_2313556; 1:500), rabbit anti-GABA (Sigma-Aldrich Cat# A2052 RRID:AB_477652; 1:2000), mouse anti-GABA (Sigma-Aldrich Cat# A0310 RRID:AB_476667, 1:500), and rabbit anti-DARPP32 (Santa Cruz Biotechnology Cat# sc-11365 RRID:AB_639000; 1:400). The secondary antibodies included goat anti-rabbit, mouse, or chicken IgG conjugated with Alexa-488, −594, or −647 (Thermo Fisher Scientific, Waltham, MA). Images were captured using a Leica SP5X white light laser confocal system with Leica Application Suite (LAS) Advanced Fluorescence 2.7.3.9723.