Mouse anti vglut1

Syt4, Mef2c,
Kif1a and Rac3 cDNA was cloned
into pcDNA3.1(−) (Thermofisher Scientific).
Tbr1^layer5 mutant cells were
transfected with Syt4, Mef2c,
Kif1a, Rac3 expression vectors and
Tbr1^wild-type were transfected with
mock empty vector using Lipofectamine 3000 (Invitrogen) for 6 hr.
Following incubation, the media was replaced by Neurobasal medium
containing B27 supplement, Penicillin/Streptomycin, 25% glucose, and
glutamax. Cultures were grown for 14 days in vitro.
After 14 days, cultures were washed 3 times with 0.5 mL 1X PBS for 5 min
each and fixed for 15 min with 4% PFA in 1X PBS at RT. Fixed cells were
washed 3 times with 0.5 mL 1X PBS and blocked in 1X PBS containing 10%
Normal Serum, 0.1% Triton X- 100 and 2% BSA for 1 hr at RT. Primary
antibodies including mouse anti-Vglut1 (1:200, Synaptic Systems) and
rabbit anti-PSD95 (1:200, Cell Signaling; excitatory synapses), rabbit
anti-Vgat (1:500, Synaptic Systems) and mouse anti-gephyrin (1:200,
Synaptic Systems; inhibitory synapses) were diluted 1:200 in blocking
solution. Cells were stained for excitatory and inhibitory synapses with
primary antibodies for 48 hr at 4°C with gentle shaking. On a
shaker, the cells were washed 3 times with 0.5 mL 1X PBS for 5 min each
and incubated with the secondary antibody for 2 hr (room temperature),
washed 3X with 1X PBS, and mounted. This experiment was repeated twice
(n = 2).

Animals were anesthetized with an intraperitoneal injection of 100 mg/kg ketamine containing 15 mg/kg xylazine. Animals were then perfused transcardially with ice-cold 1× PBS and then with 4% PFA in 1× PBS, followed by brain isolation, 1–2 h post-fixation in 4% PFA in 1X PBS at RT, cryoprotected in 30% sucrose in 1× PBS overnight at 4 °C, and cut frozen coronally on a sliding microtome at 40 μm for immunohistochemistry. All primary and secondary antibodies were diluted in 1× PBS containing 10% normal serum, 0.25% Triton X-100, and 2% BSA. The following primary antibodies were used: mouse anti-Vglut1 (1:200, Synaptic Systems; RRID: AB_887875), rabbit anti-Vgat (1:500, Synaptic Systems; RRID: AB_887871), rabbit anti-PSD95 (1:200, Cell Signaling; RRID: AB_2307331), and mouse anti-gephyrin (1:200, Synaptic Systems; RRID: AB_887717). The secondary antibodies for immunofluorescence were goat anti-rabbit IgG Alexa Fluor 488 (1:1000, Thermo Fisher; RRID: AB_143165) and goat anti-mouse Alexa Fluor 647 (1:1000, Thermo Fisher; RRID: AB_2633277). For in vivo synapse immunohistochemistry, a total of n = 10 apical dendrites were counted from each of Tbr1^wildtype and Tbr1^layer5 homozygous mutants of mixed gender. The coronal sections were pre-treated with pepsin to enhance the staining. Immunofluorescence specimens were counterstained with 1% DAPI to assist the delineation of cortical layers.