Freshly dissected cerebella from Mwk mice and wild-type littermates were fixed overnight in 4% paraformaldehyde, cryoprotected in 30% sucrose, embedded in OCT compound (TissueTek) and frozen. 10-μm sagittal sections were incubated for 30 min in 0.1 M glycine buffer (pH7.4), blocked for 1 h (10% goat serum, 0.3% Triton-X100 in PBS) and incubated with primary antibodies in block solution overnight at 4°C. The following antibodies were used: VGLUT1 (Synaptic Systems, 1:500), VGLUT2 (Synaptic Systems; 1:200), p-CaMKIV (Thr196) (Santa Cruz; 1:50) and Calbindin D28k (Synaptic Systems; 1:200). Sections were incubated with Alexa Fluor-labeled secondary antibodies (Invitrogen; 1:2000) for 3 h at room temperature before being mounted using DAPI-containing Vectashield medium (Vector Labs).
Vglut2
Manufactured by Synaptic Systems
Sourced in Germany
VGLUT2 is a protein that functions as a vesicular glutamate transporter. It is responsible for loading glutamate into synaptic vesicles, which is a crucial step in the process of glutamate-mediated neurotransmission.
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Lab products found in correlation
Vglut1, by Synaptic Systems (8 mentions)
Image lab software, by Bio-Rad (2 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Neuronal nuclei (neun), by Merck Group (2 mentions)
T4144 1vl, by Abcam (2 mentions)
Cyanine3 (cy3), by Jackson ImmunoResearch (2 mentions)
Alexa fluor labeled secondary antibody, by Thermo Fisher Scientific (1 mentions)
Calbindin d28k, by Synaptic Systems (1 mentions)
Vectashield medium, by Vector Laboratories (1 mentions)
Horseradish peroxidase, by Jackson ImmunoResearch (1 mentions)
Goat serum, by Merck Group (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Psd 95, by Thermo Fisher Scientific (1 mentions)
Nf200, by Merck Group (1 mentions)
Bassoon, by Stressgen (1 mentions)
Axio observer z1 epifluorescence microscope, by Zeiss (1 mentions)
13 protocols using vglut2
1
Immunostaining of Cerebellar Sections in Mwk Mice
2
Protein Isolation and Western Blot Analysis
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Proteins were isolated from snap‐frozen mouse vermis. Organs were homogenized in ice‐cold RIPA buffer supplemented with a complete protease inhibitor cocktail (Roche, Basel, Switzerland) and PMSF (Sigma‐Aldrich, Saint Louis, MO, USA). Samples were centrifuged for 15 minutes at 10 000 rpm, and protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Equal amounts were loaded onto SDS‐PAGE gels. After electrophoresis, the proteins were transferred to nitrocellulose membranes (Life Technologies, Carlsbad, CA, USA) and blocked with 5% non‐fat milk in Tris‐buffered saline (TBS)‐Tween. Blots were probed with primary antibodies against vGlut1 (rabbit, 1:1000, Synaptic Systems, Göttingen, Germany), vGlut2 (rabbit, 1:1000, Synaptic Systems, Göttingen, Germany) and actin (mouse, 1:5000, MP Biomedicals, Irvine, CA, USA). Secondary antibodies were conjugated with horseradish peroxidase (goat, 1:10 000, Jackson ImmunoResearch Laboratories, Suffolk, UK). Densitometric analysis was performed using ImageLab software (BioRad Laboratories, Hercules, CA, USA).
3
Immunofluorescence Imaging of Neurotransmitter Markers
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Butterfly expressing free-floating slices from the imaging experiments were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) overnight before permeabilization (0.2% Triton X-100) and blocking with 5% goat serum (Sigma) in PBS. Overnight exposure to antibodies to vGlut2 (Synaptic Systems, Goettingen, Germany, Cat. No. 135403, rabbit) and CUX1 (Santa Cruz, Dallas, TX, Cat. No. M-222, rabbit) was followed by secondary detection (4 h) using an anti-rabbit Alexa647 coupled secondary antibody (Invitrogen) all in PBS. No specific staining was observed in the absence of primary antibody. Slices were visualized with 10×, NA 0.3 and 40×, NA 0.75 objectives with a Nikon A1R confocal microscope using 488 nm and 635 nm laser lines (Coherent Scientific, Hilton, SA, Australia) for excitation of Butterfly and Alexa 647, respectively. Fluorescence was detected through band pass filters 525 ± 50 and 630 ± 50 nm).
4
Quantitative Protein Analysis in Mouse Vermis
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Proteins were isolated from snap-frozen mouse vermis. Organs were homogenized in ice-cold Ripa buffer supplemented with a complete protease inhibitor cocktail (Roche, Basel, Switzerland) and PMSF (Sigma-Aldrich, Saint Louis, MO, USA). Samples were centrifuged for 15 min at 10,000 rpm, and protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Scienti c, Waltham, MA, USA). Equal amounts were loaded onto SDS-PAGE gels. After electrophoresis, the proteins were transferred to nitrocellulose membranes (Life Technologies, Carlsbad, CA, USA) and blocked with 5% nonfat milk in Tris-buffered saline (TBS)-Tween. Blots were probed with primary antibodies against vGlut1 (rabbit, 1:1000, Synaptic Systems, Göttingen, Germany), vGlut2 (rabbit, 1:1000, Synaptic Systems, Göttingen, Germany), and actin (mouse, 1:5000, MP Biomedicals, Irvine, CA, USA). Secondary antibodies were conjugated with horseradish peroxidase (goat, 1:10 000, Jackson ImmunoResearch Laboratories, Suffolk, UK). Densitometric analysis was performed using ImageLab software (BioRad Laboratories, Hercules, CA, USA).
5
Immunohistochemical Analysis of Spinal Cord
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After the mice were transcardially fixed with 4% paraformaldehyde, the spinal cord was removed, dehydrated and embedded in OCT compound. The frozen tissue was cut in the sagittal or axial plane in 16 μm sections. For immunostaining, the sections were stained overnight at 4 °C with primary antibodies against MBP (myelin marker, 1:200, rat, Chemicon), NF200 (neuronal fiber marker, 1:200, rabbit, Sigma-Aldrich), NF200 (1:200, mouse, Sigma-Aldrich), 5-HT (serotonergic marker, 1:200, goat, ImmunoStar), VGluT2 (presynaptic marker, 1:200, guinea pig, Synaptic Systems), PSD95 (postsynaptic marker, 1:200, rabbit, Invitrogen), Hu (neuronal marker, 1:1000, human, a gift from Dr. Robert Darnell, The Rockefeller University, New York, NY, USA), Bassoon (pan-presynaptic marker, 1:200, mouse, Stressgen), NeuN (neuronal marker, 1:1000, mouse, Chemicon), c-fos (neuronal activity marker, 1:200, rabbit, Santa Cruz Biotechnology), or ChAT (1:100, goat, Chemicon). Then the sections were then incubated at room temperature for 1 h with Alexa Fluor-conjugated secondary antibodies (1:200, Invitrogen). Nuclear counterstaining was obtained using Hoechst 33342 (Molecular Probes).
6
Immunohistochemical Analysis of Mouse Brain
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Mice were deeply anesthetized with pentobarbital (200mg/kg s.c., VetOne) and transcardially perfused for 2m with ice-cold phosphate buffered saline (PBS) then for 8 min with ice-cold 4% paraformaldehyde (PFA) at a rate of 5-6ml/min. Brains were prepared as previously described [37] . Primary antibodies used: DsRed (Rabbit, 1:2000, Clontech), VGLUT1 (Guinea Pig, 1:2000, Synaptic Systems), VGLUT2 (Rabbit, 1:1000, Synaptic Systems), ChAT (Goat, 1:200, Millipore).
Secondary antibodies used (5ug/ml, Jackson ImmunoResearch): Alexa488 Donkey Anti-Goat (705-545-147), Alexa 488 Donkey Anti-Guinea Pig (706-545-148), Alexa594 Donkey Anti-Guinea Pig (706-585-148), Alexa 594 Donkey Anti-Rabbit (711-585-152), Alexa647 Donkey Anti-Rabbit (711-605-152), Alexa647 Donkey Anti-Goat (705-605-147). Images were captured using a Zeiss AxioObserver Z1 epifluorescence microscope (10x 0.45NA, 20x 0.75NA, or 63x 1.4NA objective) and Zen software. Densitometry was done with Fiji/ImageJ.
Secondary antibodies used (5ug/ml, Jackson ImmunoResearch): Alexa488 Donkey Anti-Goat (705-545-147), Alexa 488 Donkey Anti-Guinea Pig (706-545-148), Alexa594 Donkey Anti-Guinea Pig (706-585-148), Alexa 594 Donkey Anti-Rabbit (711-585-152), Alexa647 Donkey Anti-Rabbit (711-605-152), Alexa647 Donkey Anti-Goat (705-605-147). Images were captured using a Zeiss AxioObserver Z1 epifluorescence microscope (10x 0.45NA, 20x 0.75NA, or 63x 1.4NA objective) and Zen software. Densitometry was done with Fiji/ImageJ.
7
Perfusion and Immunohistochemistry of Mouse Brains
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On P12 or 60, male mice were anesthetized and perfused transcardially with saline followed by fixative (4% paraformaldehyde in borate buffer, pH 9.5). Brains were postfixed in a solution of 20% sucrose in fixative, cryoprotected overnight in 20% sucrose in 0.2M KPBS, and frozen in powdered dry ice for later processing. 20 μm thick sections were collected and used for immunohistochemical staining, as described previously [31] (link). Briefly, tissue sections were incubated overnight in blocking buffer (0.2M KPBS, 2% normal goat serum, 0.3% Triton X-100) at 4 °C followed by incubation for 72 h at 4 °C in blocking buffer containing combinations of antibodies directed against VGLUT2 (Synaptic Systems), VGAT (Synaptic Systems), or HuC/D (Life Technologies). After several rinses in KPBS, sections were incubated in blocking buffer containing a cocktail of species-specific Alexa Fluor conjugated secondary antibodies (Life Technologies). Sections were rinsed in KPBS and mounted on gel-subbed slides, and coverslipped using Fluoromount G mounting medium (Southern Biotech).
8
Antibodies for Neurochemical Analysis
9
Immunohistochemistry of Neurotransmitter Transporters
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Antibodies for the following antigens were purchased: rabbit anti-vesicular glutamate transporter 2 (VGluT2) (Synaptic Systems, Goettingen, Germany; diluted 1:500), rabbit anti-vesicular glutamate transporter 1 (VGluT1) (Synaptic Systems; diluted 1:500), rabbit anti-vesicle associated choline transporter (VAChT)(Synaptic Systems; diluted 1:250), rabbit anti-vesicular GABA transporter (VGAT) (Synaptic Systems; diluted 1:500), rabbit anti-glutamate decarboxylase 65/67 (GAD65) (Millipore Bioscience Research Reagents, Darmstadt, Germany; 1:500), and mouse anti-glutamate decarboxylase 67 (GAD67) (Millipore Bioscience Research Reagents; 1:500). For more details see Table 1 . Fluorescently conjugated secondary antibodies were purchased from Invitrogen (diluted 1:1000(Invitrogen/Life Technologies, Grand Island, NY).
10
Immunocytochemistry and Western Blot Antibody Validation
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Primary antibodies used for immunocytochemistry were all diluted 1:200, for western blotting a dilution of 1:500 was used (except for Actin which was diluted 1:100000). The Shank2 (“ppI-SAM pabSA5192”) and Shank3 antibodies (“PRC pab,” simply referred to as “Shank3” in the study and “C-term/ProSAP2/Shank3”) have been characterized previously (Bockers et al., 1999 (link); Bockmann et al., 2002 (link); Schmeisser et al., 2012 (link)). The following primary antibodies were purchased from commercial suppliers: Actin (Sigma-Aldrich Cat# A2228 RRID:AB_476697 ), Bassoon (Enzo Life Sciences Cat# ADI-VAM-PS003-F RRID:AB_11181058 ), CTIP2 (Abcam Cat# ab18465 RRID:AB_2064130 ) GAD65 (Abcam Cat# ab85866 RRID:AB_1860505 ), Homer 1b/c (Synaptic Systems GmbH Cat# 160 023, no RRID yet), GluN1 (Sigma-Aldrich Cat# G8913 RRID:AB_259978 ), Shank1 (Novus Cat# NB300-167 RRID:AB_2187584 ), SPO (Synaptic Systems GmbH Cat# 102 002 RRID:AB_887841 ), Syn1/2 (Synaptic Systems GmbH Cat# 160 003, no RRID yet), VGLUT1 (Synaptic Systems GmbH Cat# 135 304 RRID:AB_887878 ), VGLUT2 (Synaptic Systems GmbH Cat# 135 404 RRID:AB_887884 ), and VGLUT 3 (Synaptic Systems Cat# 135204, no RRID yet).
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