polyclonal guinea pig anti-vesicular glutamate transporter 1  (Synaptic Systems)

Journal: Cell Death & Disease

Article Title: The dual action of glioma-derived exosomes on neuronal activity: synchronization and disruption of synchrony

doi: 10.1038/s41419-022-05144-6

Figure Lengend Snippet: A Representative confocal images of excitatory synapses of hippocampal neurons from control (Ctr) and U87 exosome-treated (Exo) groups at DIV 4 and DIV 7–12. Neurons were identified by β3-tubulin staining (purple channel), and excitatory synapses were identified using double immunostaining with VGLUT1 (green channel)/PSD95 (red channel). Colocalization of pre- and post-synaptic markers is highlighted in yellow and corresponds to bona fide excitatory synapses. C VGLUT1/PSD95 colocalization was evaluated using Mander’s colocalization coefficient (MCC). tM1 corresponds to the fraction of VGLUT1 in compartments containing PSD95, while tM2 to the fraction of PSD95 in compartments containing VGLUT1. B Representative confocal images of inhibitory synapses identified by double immunostaining with VGAT (pre-synaptic, red channel)/GEPHYRIN (post-synaptic, green channel) under the same experimental conditions shown in ( A ). D VGAT/GEPHYRIN colocalization analysis was obtained as described in ( B ). Scale bar: 5 μm. Bar graphs show the average ± SEM of four different coverslips prepared from n = 4 distinct preparations. Unpaired Student’s t -test, ** p < 0.01. For each coverslip at least 10 dendrites were analyzed. E Upper panels : Representative traces from recordings in voltage-clamp (Black: Control, blue: GASC-479 exosome-treated) showing spontaneous synaptic events evoked by GABA or glutamate (Gray and orange color, respectively). Lower panels : Average peak currents distribution from the recordings at DIV 7–12 for control cells ( n = 3) and GASC-479 exosome-treated cells ( n = 4) in cultures with low synaptic activity. Left : GABAergic events distribution, Right : Glutamatergic events distribution (* p < 0.05 Kolmogorov–Smirnov test).

Article Snippet: Cells were then treated with a blocking solution, to saturate unspecific binding sites, composed of 10% Normal Goat Serum (Sigma-Aldrich ® , cat. no. NS02L-1ML), 0.1% Tween TM 20 Surfact-Amps TM detergent solution (Thermo Fisher Scientific) and 5% BSA at RT for 45 min. Incubation with the following primary antibodies: mouse monoclonal anti-Actin-related protein3 (Arp3; Abcam, 1:200, Cat. no. ab4967), polyclonal chicken anti-β3 tubulin (Abcam, 1:500, cat. no. ab41489), guinea pig polyclonal anti-vesicular glutamate transporter-1 (VGLUT1; Thermo Fisher Scientific, 1:2000, cat. no. AB 5905), polyclonal rabbit anti-postsynaptic density protein 95 (PSD95; Abcam, 1:500, cat. no. ab18258), mouse monoclonal anti-vesicular GABA transporter (VGAT; Synaptic Systems, 1:200, cat. no. 131011) and rabbit polyclonal anti-Gephyrin (Genetex, 1:250, cat. no. 109734) was conducted at 37 °C for 1 h. The following secondary antibodies were used for the detection: goat anti-mouse Alexa Fluor 488 (Invitrogen, Life Technologies, 1:600, cat. no. A11029), goat anti-rabbit Alexa Fluor 594 (Invitrogen, 1:600, Cat. no. A11037), donkey anti-chicken Biotin conjugated (Invitrogen, 1:600, Cat. no. SA1-72003); streptavidin Alexa Fluor 647 (Invitrogen, 1:250, Cat. no. S21374) was used for the detection of biotinylated secondary antibody.

Techniques: Staining, Double Immunostaining, Activity Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Expression of SH3 and Multiple Ankyrin Repeat Domains Protein 3 in Mouse Retina

doi: 10.3389/fncel.2022.795668

Figure Lengend Snippet: Confocal microphotographs showing the distribution of SHANK3 in synaptic. Double staining (green for SHANK3; red for Synaptophysin, PSD95 and VGluT1) appear yellowish. (a–a”) Colocalization of SHANK3 with Synaptophysin in a retinal vertical section. Fluorescence double stainings show that SHANK3 (a) and the Synaptophysin (a’,a”) is the merged image of Synaptophysin and SHANK3. (b–b”) are the locally magnified images for (a–a”) . The merged image shows that the presynaptic membrane is not labeled by SHANK3. (c–c”) Colocalization of SHANK3 with PSD95 in a retinal vertical section. Fluorescence double stainings show that SHANK3 (c) and the PSD95 (c’,c”) is the merged image of PSD95 and SHANK3. (d–d”) are the locally magnified images for (c–c”) . The merged image shows that the postsynaptic membrane is SHANK3-positive. (e–e”) Colocalization of SHANK3 with VGluT1 in a retinal vertical section. Fluorescence double stainings show that SHANK3 (e) and the VGluT1 (e’,e”) is the merged image of VGluT1 and SHANK3. Scale bar = 20 μm.

Article Snippet: Additionally, Mouse anti-PSD95 monoclonal antibody (1:500 dilution, GTX634291, GeneTex, United States) was used for labeling postsynaptic density protein 95 (PSD95), Mouse antisynaptophysin monoclonal antibody (1:500 dilution, 67864-1-Ig, Proteintech, China) was used for labeling presynaptic membrane, and Guinea pig anti-vesicular glutamate transporter 1 (VGluT1) polyclonal antibody (1:2,000 dilution, Chemicon, United States) was used for labeling glutamatergic synapses.

Techniques: Double Staining, Fluorescence, Labeling