Two to three-month-old adult mice were anesthetized and perfused transcardially with 10 mL ice-cold 1x PBS followed by 10 mL 4% paraformaldehyde solution in 1x PBS. Brains were dissected and post-fixed in 4% paraformaldehyde solution for 30 min followed by 24 h cryoprotection in 30% sucrose. 20 µm-thick sagittal AOB sections were collected on microscope slides and incubated overnight at 4°C with the following primary antibodies: Anti-VGLUT2, 1:500 (Synaptic Systems) or anti-Kirrel3, 1:100 (Neuromab). After rinsing in Tris-Buffered Saline, the appropriate secondary antibody-Alexa 488 conjugate (Molecular Laboratories) was applied at 1:500 dilution to detect the primary antibody and BS lectin at 1:1500 (Vector Laboratories) was applied along with the secondary antibody. Sections were counter-stained with Hoechst at 1:20,000 dilution (Thermo Fisher Scientific).
Anti vglut2
Manufactured by Synaptic Systems
Anti-VGLUT2 is a primary antibody that specifically binds to the Vesicular Glutamate Transporter 2 (VGLUT2) protein. VGLUT2 is a marker for glutamatergic neurons and is involved in the uptake and storage of the neurotransmitter glutamate within synaptic vesicles.
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Lab products found in correlation
Bs lectin, by Vector Laboratories (3 mentions)
Hoechst, by Thermo Fisher Scientific (3 mentions)
Anti vglut1, by Synaptic Systems (2 mentions)
Anti map2, by Merck Group (1 mentions)
Anti c fos, by Santa Cruz Biotechnology (1 mentions)
Anti tdtomato, by Takara Bio (1 mentions)
Dapi fluoromount g, by Southern Biotech (1 mentions)
Anti map2, by Santa Cruz Biotechnology (1 mentions)
Anti nestin, by Merck Group (1 mentions)
Anti glial fibrillary acidic protein, by Merck Group (1 mentions)
Apotome 2 microscope, by Zeiss (1 mentions)
Alexa fluor 488 or 594, by Thermo Fisher Scientific (1 mentions)
Anti βiii tubulin, by Fortrea (1 mentions)
Neurotrace 435 455, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Alexa 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti c fos, by Synaptic Systems (1 mentions)
Anti psd95, by Merck Group (1 mentions)
Slowfade medium, by Thermo Fisher Scientific (1 mentions)
Anti vacht, by Synaptic Systems (1 mentions)
10 protocols using anti vglut2
1
Immunohistochemical Analysis of Accessory Olfactory Bulb
2
Immunohistochemical analysis of mouse hippocampus
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Mice were terminally anesthetized, transcardially perfused with saline and 20 ml of 4% paraformaldehyde (PFA), and brains were post-fixed overnight. Coronal sections (100 μm) of the hippocampus were collected and permeabilized in 0.4% Triton in PBS (PBST) for 30 min. Sections were then blocked for 30 min with 5% horse serum in PBST and incubated overnight (4°C) with primary antibody in 5% horse serum/PBST. After extensive washing, sections were incubated with the appropriate secondary antibody conjugated with Alexa 488, 568 or 647 (Molecular Probes), for 2hrs at room temperature. They were then washed in PBST (2 × 10 min) and mounted with Dapi Fluoromount-G (SouthernBiotech). The primary antibodies used were: anti-c- fos (1:500, Santa-Cruz), anti-tdTomato (1:500, Clontech), anti-MAP2 (1:500, Sigma), anti-ABBA/Mtss1L (1:500, Millipore), anti-VGLUT2 (1:500, Synaptic Systems) and anti-beta-galactosidase (1:20, Developmental Studies Hybridoma Bank). All antibodies have been well characterized in prior studies in our laboratory, and staining was not observed when the primary antibody was omitted.
3
Multimarker Immunofluorescence Profiling
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Cells were fixed with paraformaldehyde 4% for 15 min at room temperature. After a wash in PBS, blocking was performed with BSA 5% and 0.01% Triton X-100. The following primary antibodies dilutions were used: anti-Nestin 1:200 (Millipore-Merck); anti-βIII Tubulin 1:1000 (Covance); anti-MAP2 1:200 (Santa Cruz Biotechnologies); anti-glial fibrillary acidic protein 1:800 (Sigma); anti-Synaptophysin 1 (anti-Syn1) 1:200 (Synaptic Systems); and anti-glutamatergic vesicular transporter 2 (anti-VGlut2) 1:500 (Synaptic Systems). Goat anti-mouse or anti-rabbit Alexa Fluor 488 or 594 (Life Technologies) were diluted 1:1000 in blocking solution and used as secondary antibodies. Hoechst 1:10,000 was used to counterstain nuclei, and images were acquired at Zeiss (Oberkochen, Germany) ApoTome.2 microscope with AxioVision 4.8 software.
4
Mapping Opsin-Expressing Neurons in Cortical Layers
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At the conclusion of the study, electrolytic lesions were made around the tips of the electrodes to mark the recording sites. To identify Opsin-expressing neurons, we counterstained with blue-fluorescent Nissl stain (NeuroTrace 435/455, ThermoFisher) to visualize cortical layers, and performed antibody staining (Primary antibody: AntiVGLUT2, Synaptic Systems, 1:750 dilution; Secondary antibody: Alexa Fluor™ 594, Thermo Fisher Scientific, 1:500 dilution) to discern the barrels in layer IV. Whole coronal slice (50 µm thickness) images were taken with confocal microscope (4x, Nikon).
5
Brain Immunofluorescence Staining in Mice
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Adult male mice (8–12‐week old, 3–5 mice for each genotype) were anesthetized and transcardially perfused with ice‐cold PBS containing 4% paraformaldehyde. Brains were dissected, fixed overnight at 4°C, and processed as described previously by Cho et al. (2012 (link)). Sections (20 μm) were harvested on microscope slides and incubated overnight at 4°C with the following primary antibodies: anti‐Gαo, 1:500 (MBL); anti‐olfactory marker protein (OMP), 1:500 (WAKO Chemicals); anti‐Npn2, 1:20 (R&D Systems); anti‐c‐Fos, 1:500 (Synaptic Systems); and anti‐VGlut2, 1:500 (Synaptic Systems). After rinsing in tris‐buffered saline, primary antibodies were detected with the appropriate Alexa‐488‐conjugated secondary antibody at 1:500 (Molecular Probes). Bandeiraea simplicifolia (BS) lectin, 1:1500 (Vector Laboratories) was applied with the secondary antibody.
6
Immunostaining of Cultured Motoneurons
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Cultured motoneurons were fixed with 4% paraformaldehyde for 20 min (10 min on ice, 10 min at RT), permeabilized with 1% (v/v) Triton X-100 in PBS washing three times for 5 min each time. Cultures were incubated with the following primary antibodies: anti-bassoon (mouse monoclonal, 1:250, Enzo); anti-SMN (rabbit polyclonal, 1:250, Santa Cruz); anti-vGLUT2 (rabbit policlonal, 1:250, Synaptic Systems); anti-PSD-95 (mouse monoclonal, 1:250, Millipore); anti v-AChT (rabbit polyclonal, 1:250; Synaptic Systems) diluted in 5% (w/v) BSA, 1% Triton X-100 in PBS for 1 h. After washing three times for 5 min with 1% Triton X-100 in PBS, cells were incubated for 1 h with the secondary antibody (Alexa 594 or Alexa 647-conjugated donkey anti-rabbit or anti-mouse, Invitrogen) in PBS 1X containing 0.05% Triton X-100 (1:500) and washed with PBS three times for 5 min. Finally, cells were mounted with slowfade medium (Invitrogen) on microscope slides.
7
Immunofluorescence Antibody Staining
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Immunofluorescence experiments were performed using the following primary antibodies: anti-GFP for Lphn2mVenus detection (1:1000; rabbit polyclonal; A-11122; Thermo Fisher Scientific), anti-vGlut1 (1:500; guinea pig polyclonal; 135–304; Synaptic Systems), anti-vGlut2 (1:200; guinea pig polyclonal; 135–404; Synaptic Systems), and anti-Homer1 (1:200; mouse monoclonal; 160-011; Synaptic Systems). Primary antibodies were detected using the following fluorophore-conjugated polyclonal secondary antibodies: goat anti-rabbit Alexa-Plus 488 (1:1000 A32731; Thermo Fisher Scientific), goat anti-mouse Alexa-Plus 555 (A32727; Thermo Fisher Scientific), and goat anti-guinea pig Alexa 633 (1:1000 A21105; Thermo Fisher Scientific).
8
Immunohistochemical Analysis of Accessory Olfactory Bulb
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Two to three-month-old adult mice were anesthetized and perfused transcardially with 10 mL ice-cold 1x PBS followed by 10 mL 4% paraformaldehyde solution in 1x PBS. Brains were dissected and post-fixed in 4% paraformaldehyde solution for 30 min followed by 24 h cryoprotection in 30% sucrose. 20 µm-thick sagittal AOB sections were collected on microscope slides and incubated overnight at 4°C with the following primary antibodies: Anti-VGLUT2, 1:500 (Synaptic Systems) or anti-Kirrel3, 1:100 (Neuromab). After rinsing in Tris-Buffered Saline, the appropriate secondary antibody-Alexa 488 conjugate (Molecular Laboratories) was applied at 1:500 dilution to detect the primary antibody and BS lectin at 1:1500 (Vector Laboratories) was applied along with the secondary antibody. Sections were counter-stained with Hoechst at 1:20,000 dilution (Thermo Fisher Scientific).
9
Immunohistochemical Analysis of Synaptic Markers
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Mice were transcardially perfused with 4% PFA (Sigma, cat #441244). Brains were sectioned with a cryostat (Leica CM3050 S) and incubated in the following primary antibodies: anti-VGlut1 (Synaptic Systems cat#135 303, 1:1000), anti-Vglut2 (Synaptic Systems cat#135 404, 1:2000), anti-Myelin Basic Protein (Millipore MAB384, 1:150). Sections were counterstained with DAPI (300 nM, Invitrogen D3571).
Images were acquired with a laser scanning confocal microscope (Zeiss 710) using a ×63 objective (1.4 NA, VGlut) or ×20 objective (0.8 NA, MBP). Quantitative analyses were performed on a minimum of 4-6 sections per mouse, in 3-6 mice per genotype. Analysis was performed with ImageJ software.
Images were acquired with a laser scanning confocal microscope (Zeiss 710) using a ×63 objective (1.4 NA, VGlut) or ×20 objective (0.8 NA, MBP). Quantitative analyses were performed on a minimum of 4-6 sections per mouse, in 3-6 mice per genotype. Analysis was performed with ImageJ software.
10
Immunohistochemistry of Accessory Olfactory Bulb
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Two to three-month-old adult mice were anesthetized and perfused transcardially with 10 ml ice-cold 1x PBS followed by 10 ml 4% paraformaldehyde solution in 1x PBS. Brains were dissected and post-fixed in 4% paraformaldehyde solution for 30 min followed by 24 h cryoprotection in 30% sucrose. 20 µm-thick sagittal AOB sections were collected on microscope slides and incubated overnight at 4°C with the following primary antibodies: Anti-VGLUT2, 1:500 (Synaptic Systems) or anti-Kirrel3, 1:100 (Neuromab). After rinsing in Tris-Buffered Saline, the appropriate secondary antibody-Alexa 488 conjugate (Molecular Laboratories) was applied at 1:500 dilution to detect the primary antibody and BS lectin at 1:1500 (Vector Laboratories) was applied along with the secondary antibody.
Sections were counter-stained with Hoechst at 1:20,000 dilution (Molecular Probes).
Sections were counter-stained with Hoechst at 1:20,000 dilution (Molecular Probes).
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