A2052

Cells (~ 1 × 10⁵) were cultured on a cover glass in a 12‐well plate with 700 μL of medium. The neural cells were allowed to grow to desired morphology and density before staining procedure. Cells were first washed once with PBS and fixed by 4% paraformaldehyde/4% sucrose in PBS at room temp, followed by permeabilization and DNA denaturation by 0.2% TritonX‐100 in 4 m HCl. After that, the cells were washed with PBS and blocked in 80 μL BSA (3%). The cells were incubated with anti‐FOXG1 (ab18259; Abcam), Ki‐67 (BD, 550609), Otx1/2 (ab21990; Abcam, Cambridge, MA, USA), PAX6 (ab195045; Abcam), NESTIN (BD, 561230), Nkx2.1 (MAB5460; Millipore, Darmstadt, Germany), MAP2 (M4403; Sigma, St. Louis, MO, USA), GABA (A2052; Sigma) VGAT (131011; Synaptic systems, Goettingen, Germany), SYNAPSIN (Abcam), TBR1 (ab31940; Abcam), GAT1 and Glutamate (ab1511; Millipore) in BSA (3%) at 4 °C overnight, and then conjugated with and Hoechst 33342 or DAPI. The glass slides were mounted with a cover slip before imaging.

To identify UBE3A, we used a mouse monoclonal antibody (3E5, Sigma-Aldrich, cat. no. SAB1404508, RRID:AB_10740376) raised against a peptide corresponding to mouse UBE3A isoforms 1 and 3 (a.a. 315–415) and isoform 2 (a.a. 336–436). Importantly, this immunogen shares 100% sequence identity with human UBE3A isoforms 1 (a.a. 318–417), 2 (a.a. 341–441), and 3 (a.a. 338–437). We used this antibody because we had previously verified its specificity against AS model and UBE3A KO mice [41 (link)].
To identify GABA, we used a rabbit polyclonal antibody (Sigma, St. Louis, MO; A2052, lot 052K4827) developed using GABA-BSA as the immunogen. The antibody was affinity-purified using the immunogen. It showed positive binding with GABA and GABA-KLH in a dot blot assay and negative binding with bovine serum albumin (BSA) (manufacturer’s technical information).
To identify GFAP, we used a rabbit polyclonal anti-GFAP (Chemicon, AB5804) developed against purified bovine GFAP. This antibody recognizes a 51 kDa band on immunoblot of rat spinal cord.
To identify Oligodendrocyte Transcription Factor 2 (Olig2), we used a rabbit polyclonal anti-Olig2 (Millipore, AB9610) developed using a recombinant mouse Olig-2. This antibody specifically labels cells of oligodendrocyte lineage [42 (link)], consistent with our own observations [43 (link)].

Immunohistochemistry was performed as described previously^{37 (link)}. Briefly, 10 μm thick cryosections were fixed in 4% paraformaldehyde for 20 min at room temperature and then incubated in 0.1% Triton X100/phosphate-buffered saline (PBS) for 20 min at 37 °C followed by incubation in 3% bovine serum albumin/PBS for 1 h at room temperature. The cryosections were then incubated with the following primary antibodies for 1 day at 4 °C: rabbit polyclonal antibody against α7-nAChR (sc-5544, 1:20; Santa Cruz Biotechnology), rabbit polyclonal antibody against GAD65/GAD67 (ab11070, 1:200; Abcam), rabbit polyclonal antibody against GABA (A2052, 1:100; Sigma, St. Louis, MO, USA), and goat polyclonal antibody against Brn3a (sc-31984, 1:40; Santa Cruz Biotechnology). The secondary antibodies (all from Invitrogen-Molecular Probes, Eugene, OR, USA) were Alexa Fluor 555-conjugated donkey anti-rabbit IgG antibody (A31572, 1:1,000), Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (A11070, 1:500), and Alexa Fluor 488-conjugated donkey anti-goat IgG antibody (A11055, 1:500). The sections were finally counterstained with the nucleic acid stain Hoechst 33258 (H3569, 1:2,000; Invitrogen-Molecular Probes) in PBS and imaged using a laser scanning confocal microscope (TCS SP8; Leica Microsystems, Heidelberg, Germany).