Glutamate antibody

Details for Product No. ABIN617901, Supplier: Log in to see
Antigen
  • GRIN1
  • NMDAR1
  • glutamate receptor, ionotropic, N-methyl D-aspartate 1
  • GRIN1
Reactivity
Chemical
11
Host
Mouse
6
5
Clonality
Monoclonal
Application
Immunoelectron Microscopy (IEM), Immunocytochemistry (ICC), Immunofluorescence (IF), Immunohistochemistry (IHC)
7
6
3
2
1
Options
Supplier
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Supplier Product No.
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'Independent Validation' Badge
Antigen glutamate
Lot Number 136026
Method validated Immunofluorescence
Positive Control Spinal cord
Negative Control No primary antibody
Notes ABIN617901 works for immunofluorescence on free-floating vibratome sections.
Primary Antibody ABIN617901
Secondary Antibody anti-mouse Alexa Fluor 488 conjugated antibody (Thermo Fisher Scientific, R37120, lot 1073084)
Protocol
  • Perfuse mouse spinal cord at P60 with 1% paraformaldehyde and 2% glutaraldehyde in 0.1M phosphate buffer pH7.4.
  • Post-fix spinal cord blocks for in the same fixative for 3h.
  • Wash blocks 4x5 min in PBS.
  • Cut blocks with a vibratome (Leica, VT1000 S) into 50µm thick transverse sections.
  • Block free-floating sections in 0.01M PBS containing 5% normal goat serum (Sigma Aldrich, G9023, lot SLBM5884V) for 30min at RT.
  • Incubate section with primary mouse anti-glutamate antibody (antibodies-online, ABIN617901, lot 22523) diluted 1:500 in blocking solution ON at 4°C.
  • Wash sections 4x 5min in 0.01M PBS.
  • Incubate sections with secondary anti-mouse Alexa Fluor 488 conjugated antibody (Thermo Fisher Scientific, R37120, lot 1073084) diluted 1:500 in 0.1M PBS for 1h at RT.
  • Wash sections 4x 5min in 0.01M PBS.
  • Mount sections on a coverslip using Vectashield mounting medium (Vector Labs, H-1000, lot ZB0901).
  • Image acquisition with a laser scanning confocal microscope (Leica TCS SP5).
Experimental Notes
  • In order to exclude any non-specific signal due to the secondary antibody one negative control slice without the primary antibody was processed for each experiment. In this case the incubation ON at 4°C was performed in diluent solution only.
  • We tested ABIN617901 at 1:500 and 1:1000 dilutions with and without 0.1% Triton X-100.
Validation Images
Immunofluorescence validation image for anti-Glutamate antibody (ABIN617901) IF staining of P60 free-floating mouse spinal cord transversal 50µm thick sections wi...
'Independent Validation' Badge
Antigen glutamate
Lot Number 136026
Method validated Electron Microscopy
Positive Control Rat spinal cord
Negative Control no primary antibody control, superior cervical ganglion
Notes ABIN617901 works for electron microscopy immunogold labeling on cryosubstituted/epoxy sections at a dilution of 1:10.
Primary Antibody ABIN617901
Secondary Antibody goat anti-mouse secondary antibody coupled to colloidal gold particles
Protocol
  • Rat Spinal cord:
    • Perfuse rat spinal cord with glutaraldehyde (0.1%), formaldehyde (4%), and picric acid (0.2%) in 0.12 M sodium phosphate buffer, pH7.4.
    • Cut spinal cord blocks with a vibratome (Leica, VT 1000 S) into 200µm thick transverse sections.
    • Slam sections to a polished copper block cooled with liquid N2 in a MM80E cryofixation apparatus (Reichert).
    • Transfer sections to 0.5% uranyl acetate dissolved in anhydrous methanol (-90°C) in a freeze-substitution apparatus (Leica, CS Auto).
    • Raise temperature stepwise to -50°C.
    • Infiltrate samples with Lowicryl HM20 resin (Chemische Werke Lowi, Electron Microscopy Sciences, 14340) and photopolymerize by UV light.
    • Cut 70-80nm ultrathin sections with an ultramicrotome (Leica EM UC6).
    • Etch sections with a saturated solution of NaOH in absolute ethanol for 2-3sec.
    • Rinse sections with double-distilled water.
    • Incubate sections in 0.1% sodium borohydride and 50mM glycine in 5mM Tris buffer containing 0.1% NaCl and 0.1% Triton X-100 (TBNT) for 10min at RT.
    • Incubate sections for 10min in 2% bovine serum albumin (BSA) in TBNT for 10min at RT.
    • Incubate sections with primary mouse anti-glutamate antibody (antibodies-online, ABIN617901, lot 22523) diluted 1:10 in TBNT containing 2% BSA ON at RT.
    • Rinse sections several times with TBNT containing 2% BSA for 10min at RT.
    • Incubate sections with secondary goat anti-mouse secondary antibody antibody coupled to 10nm (British BioCell International, EM.GAM10, lot 7356) or 20nm colloidal gold particles (British BioCell International, EM.GAM20, lot 008512) diluted 1:20 in TBNT containing 2% BSA and 0.05% polyethylene glycol for 2h at RT.
    • Rinse sections several times in double-distilled water.
    • Counterstain sections with uranyl acetate and lead citrate.
    • Image acquisition with JEM-1010 transmission electron microscope (Jeol) equipped with a sidemounted Mega View III CCD camera (Olympus Soft Imaging System).
  • Mouse Superior cervical ganglion:
    • Mouse superior cervical ganglia (SCGs) were dissected out and postfixed in paraformaldehyde 1% + glutaraldheyde 2% for 4h.
    • Wash SCGs severayl times in PB 0.2 M SCGs.
    • Stain SCGs with osmium ferrocyanide at for 1h at 4°C.
    • Dehydrate SCGs in increasing concentrations of acetone: acetone 30%for 15min, acetone 60% 15min, acetone 90% (15 min), acetone 100% 15min, and acetone 100% for 30min.
    • Prepare Spurr resin: 10g vinyl cyclohexene dioxide (ERL 4221; Electron Microscopy Sciences, 15004), 6g diglycidyl ether polypropylene glycol (DER; Electron Microscopy Sciences, 13010), 26g nonenyl succinic anhydride (NSA; Electron Microscopy Sciences, 19050), 0.4g dimethylamino ethanol (DMAE; Electron Microscopy Sciences, 13300).
    • Transfer SCGs to acetone 100% Spurr resin 1:1 for 30min.
    • Transfer SCGs to acetone 100% Spurr resin 1:2 for 30min.
    • Transfer SCGs to Spurr resin ON at RT.
    • Embed SCGs in polyethylene capsules in Spurr resin for 24h at 70°C.
    • Cut 70-80nm ultrathin sections with an ultramicrotome (Leica EM, UC6) and collect them on pre-cleaned uncoated nickel grids.
    • Treat SCG ultrathin sections with a saturated aqueous solution of sodium metaperiodate for 3min at RT.
    • Rinse sections in 1% Triton X-100 in Tris buffered saline (TBS) 0.5M.
    • Incubate sections in 10% goat normal serum in TBS for 1h at RT.
    • Incubate grids on drops of primary antibody anti-glutamate (1:10) in TBS ON at RT.
    • Rinse grids in TBS.
    • Incubate grids with secondary goat anti-mouse secondary antibody coupled to 10nm (British BioCell International, product EM.GAM10, lot 7356) or 20nm colloidal gold particles (British BioCell International, productEM.GAM20, lot 008512) diluted 1:15 in TBS for 1h at 37°C.
    • Transfer grids into drops of 2.5% glutaraldehyde in cacodylate buffer 0.05M.
    • Wash grids in double-distilled water.
    • Counterstain SCG sections with lead citrate for 10min at RT. Image acquisition with JEM-1010 transmission electron microscope (Jeol) equipped with a side mounted Mega View III CCD camera (Olympus Soft Imaging System).
Experimental Notes As negative controls we incubated one slice ON with the blocking solution and then processed it with the secondary antibody and performed immunogold labeling on the mouse superior cervical ganglion that does not contain glutamatergic synapses.
Validation Images
Electron Microscopy validation image for anti-Glutamate antibody (ABIN617901) A: The mouse superior cervical ganglion (SCG) contains cholinergic synapses and thus ...
Immunogen Glutamate
Isotype IgG2a
Cross-Reactivity Cat (Feline), Rat (Rattus)
Characteristics The Glutamate Antibody was raised to glutamate coupled to KLH with glutaraldehyde. The antibody produces strong labeling of glutamate at dilutions of 1/2,000 - 1/4,000 using biotin-streptavidin/HRP technique. Glutamate tissue staining is completely eliminated by preincubation with glutamate conjugate at concentrations of 100 μg conjugate per mL of diluted antiserum. Aspartate and glutamine conjugates could not significantly inhibit tissue staining. The following amino acids were tested for cross reactivity using an enzyme-linked immunoassay method. Wells were coated with Glu-Glut-Btg at 1 μg per mL. Amino acids and conjugate were added at concentrations from 10 μg to 1 ng per mL. The Glutamate antibody was added to wells at 1 μg per mL. These amino acids were found to have cross reactivity at less than 1 % : Beta-alanine, L-alanine, L-aspartic acid, L-glutamic acid, Glycine, Taurine and L-tyrosine.
Purification Whole Serum
Target Type Chemical
Research Area Signaling, Metabolism, Amino Acids
Restrictions For Research Use only
Format Liquid
Buffer Contains ≤ 0.09 % sodium azide
Preservative Sodium azide
Precaution of Use This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
Storage 4 °C/-20 °C
Storage Comment After reconstitution, use immediately or refrigerate at 2 - 8 °C up to 2 days. For long-term storage aliquot antibody and freeze at -15 °C or lower. Avoid repeated freeze/thaw cycles.
Supplier Images
Immunohistochemistry (IHC) image for anti-Glutamate antibody (ABIN617901) IHC image of neurons in rat cortex.
Product cited in: Colom, Castaneda, Aleman, Touhami: "Memantine protects cholinergic and glutamatergic septal neurons from Aβ1-40-induced toxicity." in: Neuroscience letters, Vol. 541, pp. 54-7, 2013 (PubMed).

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Colom, Castañeda, Bañuelos, Puras, García-Hernández, Hernandez, Mounsey, Benavidez, Lehker: "Medial septal beta-amyloid 1-40 injections alter septo-hippocampal anatomy and function." in: Neurobiology of aging, Vol. 31, Issue 1, pp. 46-57, 2009 (PubMed).

Uematsu, Baba, Kake, Ikenaga, Moon, Miyai, Yoshida: "Central mechanisms underlying fish swimming." in: Brain, behavior and evolution, Vol. 69, Issue 2, pp. 142-50, 2007 (PubMed).

Park, Himes, Stieg, Tessler, Fischer, Snyder: "Neural stem cells may be uniquely suited for combined gene therapy and cell replacement: Evidence from engraftment of Neurotrophin-3-expressing stem cells in hypoxic-ischemic brain injury." in: Experimental neurology, Vol. 199, Issue 1, pp. 179-90, 2006 (PubMed).

Morales, Sampogna, Rampon, Luppi, Chase: "Brainstem glycinergic neurons and their activation during active (rapid eye movement) sleep in the cat." in: Neuroscience, Vol. 142, Issue 1, pp. 37-47, 2006 (PubMed).

Garrido Sanabria, Castañeda, Banuelos, Perez-Cordova, Hernandez, Colom: "Septal GABAergic neurons are selectively vulnerable to pilocarpine-induced status epilepticus and chronic spontaneous seizures." in: Neuroscience, Vol. 142, Issue 3, pp. 871-83, 2006 (PubMed).

Morris, König, Shimizu, Jobling, Gibbins: "Most peptide-containing sensory neurons lack proteins for exocytotic release and vesicular transport of glutamate." in: The Journal of comparative neurology, Vol. 483, Issue 1, pp. 1-16, 2005 (PubMed).

Colom, Castaneda, Reyna, Hernandez, Garrido-Sanabria: "Characterization of medial septal glutamatergic neurons and their projection to the hippocampus." in: Synapse (New York, N.Y.), Vol. 58, Issue 3, pp. 151-64, 2005 (PubMed).

Vadivelu, Platik, Choi, Lacy, Shah, Qu, Holekamp, Becker, Gottlieb, Gidday, McDonald: "Multi-germ layer lineage central nervous system repair: nerve and vascular cell generation by embryonic stem cells transplanted in the injured brain." in: Journal of neurosurgery, Vol. 103, Issue 1, pp. 124-35, 2005 (PubMed).

Nagayama, Kimura, Araki, Aonuma, Newland: "Distribution of glutamatergic immunoreactive neurons in the terminal abdominal ganglion of the crayfish." in: The Journal of comparative neurology, Vol. 474, Issue 1, pp. 123-35, 2004 (PubMed).

Ando, Kuwasawa: "Neuronal and neurohormonal control of the heart in the stomatopod crustacean, Squilla oratoria." in: The Journal of experimental biology, Vol. 207, Issue Pt 26, pp. 4663-77, 2004 (PubMed).

Lee, Kim, Valentino, Waterhouse: "Glutamatergic afferent projections to the dorsal raphe nucleus of the rat." in: Brain research, Vol. 963, Issue 1-2, pp. 57-71, 2003 (PubMed).

Tong, Kirchgessner: "Localization and function of metabotropic glutamate receptor 8 in the enteric nervous system." in: American journal of physiology. Gastrointestinal and liver physiology, Vol. 285, Issue 5, pp. G992-G1003, 2003 (PubMed).

Korada, Zheng, Basilico, Schwartz, Vaccarino: "Fibroblast growth factor 2 is necessary for the growth of glutamate projection neurons in the anterior neocortex." in: The Journal of neuroscience : the official journal of the Society for Neuroscience, Vol. 22, Issue 3, pp. 863-75, 2002 (PubMed).

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F -Tsukamoto, Kuwasawa: "Neurohormonal and glutamatergic neuronal control of the cardioarterial valves in the isopod crustacean Bathynomus doederleini." in: The Journal of experimental biology, Vol. 206, Issue Pt 3, pp. 431-43, 2002 (PubMed).

Delgado, Oyola, Miller: "Localization of GABA- and glutamate-like immunoreactivity in the cardiac ganglion of the lobster Panulirus argus." in: Journal of neurocytology, Vol. 29, Issue 8, pp. 605-19, 2001 (PubMed).

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