Rabbit anti vgat

40 μm spinal cord sections were washed in PBS (3x 5 min) at room temperature and afterwards blocked in blocking solution for 30 min. Then, sections were incubated in blocking solution containing the primary antibodies or lectin in a humidified chamber for two nights at 4°C and washed in PBS containing 0.1% Triton X 100 (3x 5 min) and secondary antibody was applied in blocking solution over night at 4°C. The next day, sections were washed in PBS (3x 5 min) and mounted in Mowiol. The following antibodies and lectins were used for stainings: rabbit anti-vGAT (1:1000, Synaptic Systems), rabbit anti-vGlut1 (1:1000, Synaptic Systems), biotinylated Wisteria floribunda agglutinin (WFA) (1:200, Sigma-Aldrich (L1516). vGAT and vGlut1 stainings were carried out on consecutive sections.

Selected sections at the level of the dDG were processed using the MOM kit as described above. Sections were incubated overnight at RT in a solution containing rabbit anti-GFP (1:2000; Invitrogen), guinea pig anti-VGLUT2 (1:5000; Millipore) and mouse anti-GAD65 (1:100; Millipore) or mouse anti-GFP (1:100; Invitrogen), guinea pig anti-VGLUT2 (1:5000; Millipore) and rabbit anti-VGAT (1:1000; Synaptic System) diluted in MOM diluent. After several rinses in KPBS, they were incubated for 2 h in Alexa488-conjugated donkey anti-rabbit IgG (1:200; Invitrogen), Cy5-conjugated donkey anti-guinea pig (1:100; Jackson ImmunoResearch Laboratories, Inc.), and Cy3-conjugated donkey anti-mouse (1:100; Jackson ImmunoResearch Laboratories, Inc.) or Alexa488-conjugated donkey anti-mouse IgG (1:200; Invitrogen), Cy5-conjugated donkey anti-guinea pig (1:100; Jackson ImmunoResearch Laboratories, Inc.), and Cy3-conjugated donkey anti-rabbit (1:100; Jackson ImmunoResearch Laboratories, Inc.) diluted in MOM diluent. After several rinses in KPBS, all sections were then mounted on superfrost-coated slides, dried overnight at RT and coverslipped with Fluoromount. The specimens were analyzed with confocal microscope (Zeiss).

Cells were fixed for 15’ with 4%PFA/4%sucrose in PBS, washed and permeabilized with Triton-X100 (0.25%) in TBS. For TAU aggregates, 1%Triton-X100 was added to the fixative to remove monomeric proteins. After 30’ blocking, cells were incubated overnight at 4°C with following primary antibodies: mouse anti-β3 tubulin, mouse anti-PAX6 (both Covance), chicken anti-MAP2 (Aves), rabbit anti-Tbr1, rat anti-Ctip2, mouse anti-Nestin (all Abcam), rabbit anti-vGlut2, rabbit anti-vGAT (both Synaptic Systems), mouse anti-OCT4, mouse anti-HuC/D (both Invitrogen), mouse anti-Nanog, mouse anti-RD4 (both Millipore), mouse anti-AT8 (Innogenetics) or AT8 conjugated with Alexa 568. The next day, cells were washed and incubated for 1 hour at RT with Alexa secondary antibodies (Life Technologies). DAPI was used to stain the nuclei. Images were taken with OPERA or CV7000 high content readers or the Leica fluorescence microscope.

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).

The following primary reagents were used: chicken anti-PV (1:500, Synaptic Systems, Göttingen, Germany, 195006), biotinylated Wisteria floribunda agglutinin (WFA, 1:1000, Vector laboratories B-1355, Burlingame, CA, USA), rabbit anti-Bcan [32 (link),33 (link)] (1:1000), guinea pig anti-vGluT1 (1:1500, Synaptic Systems, 135304), mouse anti-Pcan (1:500, The Developmental Studies Hybridoma Bank, Iowa City, IA, USA, 3F8-c), mouse anti-Vcan (1:500, The Developmental Studies Hybridoma Bank, 12C5-c), rabbit anti-vGAT (1:500, Synaptic Systems), mouse anti-Iba1 (1:1000, Millipore, Boston, MA, USA, MABN92) and rabbit anti-Iba1 (1:1000, Wako, Richmond, VA, USA, 019-19741).
As secondary antibodies, we used Alexa Fluor 405 goat anti-chicken IgG (1:500, Abcam, Cambridge, UK, ab175675), Alexa Fluor 647 goat anti-chicken IgG (1:1000, Invitrogen, Waltham, MA, USA, A-21449), streptavidin Alexa Fluor 405 conjugated (1:1000, Invitrogen, S32351), Alexa Fluor 647 goat anti-rabbit IgG (1:1000, Invitrogen, S32351), Alexa Fluor 405 donkey anti-guinea pig IgG (1:500, Sigma Aldrich, St. Louis, MO, USA, SAB4600230), Alexa Fluor 647 goat anti-mouse IgG (1:1000, Invitrogen, A21236), Alexa Fluor 405 donkey anti-rabbit IgG (1:500, Abcam, ab175651), Alexa Fluor 488 goat anti-rabbit IgG (1:200, Abcam, ab150077), and Alexa Fluor 546 goat anti-guinea pig IgG (1:200, Invitrogen, A11074).