Guinea pig anti vglut1

For immunofluorescence experiments, 40–50 μm floating sections were collected in 1× PBS, then rinsed once in 1× PBT (PBS + 1% Triton 100×), and incubated overnight at 4 °C in a cocktail of primary antibodies that were diluted in 1× PBT + 20% normal donkey serum. After the overnight incubation, sections were rinsed three times with 1× PBT for 10 min and incubated for 1.5 h in the corresponding secondary antibodies (1:500, Jackson-ImmunoResearch or Invitrogen). Cerebellar sections were then washed three times with 1× PBT for 10–15 min, incubated with DAPI, and mounted in Poly-aquamount (Polysciences). The following primary antibodies were used: rabbit anti-IBA1 (1:200, Wako, # 019-19741), mouse anti-CALB (calbindin, 1:200, Sigma-Aldrich, #C9848), rat anti-CD68 (1:200, Bio-Rad, #MCA1957), Lycopersicon esculentum(Tomato)-Lectin (1:200, Sigma-Aldrich, #L0401), guinea-pig anti-VGLUT1 (1:800, Synaptic Systems, #135404), rabbit anti-phosphorylated S6R (1:200, Cell Signaling, #2211), rabbit anti-phosphorylated AKT (1:200, Cell Signaling, #8112), rabbit anti-TFEB (1:200, Bethyl Laboratories, A303-673A), rabbit anti-FASN (1:200, Cell Signaling, #3180), rabbit anti-pyruvate dehydrogenase E 1 alpha (PDE) (1:200, GeneTex, # GTX104015), rat anti-CD107a (LAMP-1) (1:200, BioLegend, # 121602).

Cultures (DIV 18–20) were washed with phosphate-buffered saline (PBS) solution, fixed with 4% paraformaldehyde, and processed as previously described (Chang et al., 2014 (link)). Primary antibodies were used as follows: rabbit anti-GFP (Abcam; Cat# Ab6556; 1:1,000 dilution); mouse anti-VGAT (Synaptic Systems; Cat# 131 011; 1:1,000 dilution); guinea pig anti-VGLUT1 (Synaptic Systems; Cat# 135 304; 1:4,000 dilution). Secondary antibodies were used as follows: Alex Fluor 488 donkey anti-rabbit (Jackson Immuno Research; Cat# 711-545-152; 1:500 dilution); Rhodamine Red donkey anti-guinea pig (Jackson Immuno Research; Cat# 706-295-148; 1:500 dilution); Alexa Fluor 647 donkey anti-mouse (Jackson Immuno Research; Cat# 715-605-151; 1:500 dilution).
Images were captured on an inverted microscope with 60× objective (water, n/a 1.2) using a CCD camera (Princeton Instruments) and mercury lamp illumination under control of MetaMorph Software (Molecular Devices). Colocalization of background-subtracted images was analyzed in ImageJ using the Pearson’s coefficient (r) of the full images determined by the JACoP colocalization plug-in (Bolte and Cordelières, 2006 (link)).

After 21 days in culture, neurons were fixed with 4% w/v paraformaldehyde (Sigma-Aldrich) for 10 min at room temperature and processed as described previously^{52 (link)}. For synapse detection, we used the antibodies against vesicular glutamate transporter VGLUT1 (guinea pig anti-VGLUT1, 1:500, Synaptic Systems, #135304), postsynaptic density protein PSD95 (mouse anti-PSD95, 1:500, Millipore, MAB1598), vesicular GABA transporter VGAT (guinea pig anti-VGAT, 1:500, Synaptic Systems, #131103), and gephyrin (mouse anti-gephyrin, 1:500, Synaptic Systems, #147011). For cell type identification, the antibodies against GABA (rabbit anti-GABA, 1:2000, Sigma-Aldrich, A2052), parvalbumin (chicken anti-parvalbumin, 1:500, Synaptic Systems, #195006) and aggrecan (rabbit anti-aggrecan, 1:500, Millipore, AB1031; mouse anti-aggrecan, 1:500, R&D Systems, MAB1220) were applied. To enable the immunofluorescence detection, the following secondary antibodies were used: anti-mouse IgG Alexa 488 (1:250, Jackson Immuno, #715-545-150), anti-mouse IgG Alexa 594 (1:500, Jackson Immuno, #715-545-151), anti-mouse IgG Alexa 647 (1:500, Jackson Immuno, #115-605-003), anti-rabbit IgG Alexa 594 (1:500, Jackson Immuno, #711-585-152), anti-guinea pig IgG Alexa 647 (1:500, Jackson Immuno, #706-605-148), and anti-chicken IgY Alexa 488 (1:250, Jackson Immuno, #103-545-155).

Mice were anesthetized with an i.p. injection of Tribromoethanol (250 mg/Kg body weight) and transcardially perfused with ice-cold 0.1 M phosphate buffer (PB) (pH 7.4). Brains were removed and rapidly frozen in OCT using dry ice and 100% ethanol. 5 μm-thick coronal sections of the brainstem were obtained using a cryostat, thaw mounted on microscope slides (Fisherbrand Superfros Plus, Fisher Scientific) and air-dried for 15 min. Sections were fixed by immersion in ice-cold 95% ethanol at 4°C for 30 min followed by acetone for 1 min at RT. Prior to the staining, sections were washed in 0.1 M PB and incubated 20 min at RT in 0.1 M PB blocking solution containing 2% normal goat serum (NGS) and 0.2% Triton X-100. Afterward, sections were incubated at 4°C overnight with the primary antibodies diluted in the same blocking solution (1:100, Rabbit anti-CAST, and 1:100 Rabbit anti-ELKS, T99; 1:100, polyclonal Guinea pig anti-Vglut1, Synaptic Systems). Subsequently, sections were rinsed 3×10 min in 0.1 M PB and incubated with the secondary antibodies diluted in 0.1 M PB containing 0.1% Triton X-100 for 2h at RT 1:200 (Cy 2 AffiniPure goat anti-rabbit IgG (H+L), Jackson Immunoresearch; 1:200, Alexa Fluor® 647-conjugated AffiniPure donkey anti-guinea pig, Jackson Immunoresearch). After 3×10 min in 0.1 M PB washed sections were mounted using Fluoromount-G® mounting medium (SouthernBiotech).

Synaptic density of cultured neurons was measured as we previously described^{19 (link)}. Cultured neurons were fixed in 4% paraformaldehyde for 30 min followed by washing using PBS. Fixed neurons were blocked in 5% goat serum for 30 min at room temperature. Postsynaptic density protein 95 (PSD-95), vesicular glutamate transporter 1 (vGlut1) and MAP2 were labelled by rabbit anti-PSD95 (Cell Signaling, #3450, 1:200), guinea pig anti-vGlut1 (Synaptic System, #135304, 1:400) and mouse anti-MAP2 (Sigma-Aldrich, M4403, 1:400) followed by goat anti-rabbit IgG conjugated with Alexa 594 (Invitrogen, A11037, 1:500), goat anti-guinea pig IgG conjugated with Alexa 647 (Invitrogen, A21450, 1:500) and goat anti-mouse IgG conjugated with Alexa 488 (Invitrogen, A11029, 1:500), respectively. Images were captured by using a Nikon confocal microscope followed by three-dimensional reconstruction by using Nikon-Elements Advanced Research software. The synapses were defined by colocalization of vGlut1 and PSD95. Dendritic segments between 30 and 60 nm from the soma were used for the analysis.

Synaptic density of cultured neurons was measured by counting PSD95 and vGlut1-labelled clusters attaching to neuronal dendrites and presented as the number of synapses per micron of dendrite as previously described67 (link). Briefly, after fixation in 4% paraformaldehyde for 30 min, neurons were blocked in 5% goat serum for 30 min at room temperature. PSD95, vGlut1 and MAP2 were labelled by rabbit anti-PSD95 (Cell Signaling, #3450, 1:200), guinea pig anti-vGlut1 (Synaptic System, #135304, 1:400) and mouse anti-MAP2 (Sigma-Aldrich, M4403, 1:400) followed by goat anti-rabbit IgG conjugated with Alexa 594 (Invitrogen, A11037, 1:500), goat anti-guinea pig IgG conjugated with Alexa 647 (Invitrogen, A21450, 1:500) and goat anti-mouse IgG conjugated with Alexa 488 (Invitrogen, A11029, 1:500), respectively. Images were collected under a Nikon confocal microscope followed by three-dimensional reconstruction by using Nikon-Elements advanced Research software68 (link). The synapses were defined by colocalization of vGlut1 and PSD95. Dendritic segments between 20 and 50 μm from the soma were used for the analysis69 (link).