Goat serum

Neuron staining and confocal immunofluorescence microscopy analysis were performed as described previously ^{14 (link)}. Briefly, to label cell surface proteins, iPSC-derived GABAergic neurons on the chamber slides were fixed with 4% paraformaldehyde in DPBS for 10 min. We then blocked with 10% goat serum (ThermoFisher, catalog #: 16210064) in DPBS for 0.5 h, and without detergent permeabilization, incubated with 100 μL of appropriate primary antibodies against the GABA_A receptor $\mathrm{\alpha }1$ subunit (Synaptic Systems, Goettingen, Germany, catalog #: 224203) (1:250 dilution), $\mathrm{\beta }2/3$ subunit (Millipore, catalog #: 05–474) (1:250 dilution), or $\mathrm{\gamma }2$ subunit (Synaptic Systems, Goettingen, Germany, catalog #: 224003) (1:250) diluted in 2% goat serum in DPBS, at room temperature for 1 h. Then the neurons were incubated with Alexa 594-conjugated goat anti-rabbit antibody (ThermoFisher, catalog #: A11037), or Alexa 594-conjugated goat anti-mouse antibody (ThermoFisher, catalog #: A11032) (1:500 dilution) diluted in 2% goat serum in DPBS for 1 h. Afterward, the chamber slides were mounted using fluoromount-G (VWR, catalog #: 100502–406) and sealed. An Olympus IX-81 Fluoview FV3000 confocal laser scanning system was used. A 60× 1.40 numerical aperture oil objective was used to collect high-resolution images using FV31S-SW software. The images were analyzed using ImageJ software ^{69 (link)}.

0:5×10⁵ cells were seeded on poly-Lysine coated high-precision glass coverslips (18 mm round, #1.5) in 12-well culture plates one day prior to transfection. Transfection was performed as described for flow cytometry experiments. A total amount of 200 ng DNA (20 ng synTFs and 180 ng ssDNA) was used for transfection experiments. PEI was scaled to 12-well plate volume of 100 μL total transfection mix. 48 h post transfection, cells were washed with 1x PBS, fixed with 2% PFA (Fisher Scientific) and blocked for 30 min with 10% Goat serum (VWR) in 1x PBS after washing. Immunodetection was performed using anti-HA-tag (6E2) mouse monoclonal antibody (Cell Signaling) and anti-GFP (D5.1) rabbit monoclonal antibody (Cell Signaling) 1:200 in 1%BSA/PBS overnight. Cells were washed with 0.1% Triton X-100 and incubated with anti-mouse IgG Alexa Fluor 488 (#4408, Cell Signaling) and anti-rabbit IgG Alexa Fluor 647 antibodies 1:1000 in 1%BSA/PBS for 1 h. After washing with 0.1% Triton X-100, nuclei were stained with 2 μg/mL Hoechst-33342 (Thermo Fisher Scientific) and mounted on glass slides using Prolong Gold Antifade (Thermo Fisher Scientific). Image acquisition was performed at least 16 h after mounting coverslips on glass slides.