Gad65 67

Manufactured by Merck Group

Sourced in United States

GAD65/67 is a laboratory reagent used in the detection and quantification of the glutamic acid decarboxylase (GAD) enzyme. GAD is an important enzyme involved in the production of the neurotransmitter gamma-aminobutyric acid (GABA). This reagent can be used in various biochemical and immunological assays to measure GAD levels in biological samples.

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Rabbit anti-GAD65/67 (5 citations) GAD65 (3 citations) Anti‐GAD65‐67 (3 citations) Mouse anti-Gad65/67 (2 citations)

8 protocols using gad65 67

Quantitative Western Blot Analysis

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To acquire western blot data, proteins were separated using a 10 − 15 % sodium dodecyl sulfate-polyacrylamide gel and transferred onto polyvinylidene fluoride membranes. The membranes were incubated with blocking buffer [5 % nonfat dry milk in phosphate-buffered saline containing 0.05 % Tween-20 (PBST)] for 1 h at room temperature. They were then incubated with the indicated antibodies overnight at 4 °C and washed three times with PBST. The membranes were incubated with corresponding secondary antibodies for 1 h at room temperature. After washing with PBST, proteins were detected with enhanced chemiluminescence solution (Pierce) and LAS-4000 (Fujifilm, Tokyo, Japan). The intensity of each band was determined using an analysis system (Multi Gauge version 3.0, Fujifilm, Tokyo, Japan). The membranes were incubated with antibodies to glutamate acid decarboxylase 65/67 (GAD 65&67) (1:500; Millipore, Temecula, CA, USA), glutamate transporter (GT) (1:4000; Abcam, Cambridge, UK), and β-actin (1:1000; Sigma-Aldrich).

Lee J.E., Jeong D.U., Lee J., Chang W.S, & Chang J.W. (2016). The effect of nucleus basalis magnocellularis deep brain stimulation on memory function in a rat model of dementia. BMC Neurology, 16, 6.

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Western Blot Analysis of Hippocampal Proteins

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Total protein extracts were prepared from mice hippocampi. Protein isolation was done using RIPA buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1.5 mM MgCl₂, 100 mM NaF, 1 mM Na₃VO₄, 1 mM EGTA, 1% Triton X-100, 10% Glycerol) with protease inhibitor cocktail (Roche). For western blotting, 20 μg of total protein were separated on 10% SDS-PAGE and transferred to a PVDF membrane. Membranes were blocked and antibodies were diluted in 5% dry milk/TBST. Primary antibodies used were: Gad65/67 (Millipore) 1:500 dilution, Wwox (using affinitypurified anti-WWOX rabbit polyclonal primary antibodies developed in our laboratory) 1:1000 dilution, HRP conjugated secondary antibodies were used followed by chemiluminescence (GE Healthcare). Actin was used as the protein loading control, and it was detected using monoclonal anti-actin antibody (Sigma) 1:5000 dilution and HRP conjugated anti-mouse secondary antibody 1:5000 dilution.

Hussain T., Kil H., Hattiangady B., Lee J., Kodali M., Shuai B., Attaluri S., Takata Y., Shen J., Abba M.C., Shetty A.K, & Aldaz C.M. (2018). Wwox deletion leads to reduced GABA-ergic inhibitory interneuron numbers and activation of microglia and astrocytes in mouse hippocampus. Neurobiology of disease, 121, 163-176.

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Western Blot Analysis of Neuronal Markers

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Brain tissues were homogenized in the sample lysis buffer (62.5 mM Tris-HCl pH 6.8, 2 % SDS, 0.5 % NP-40, 5 mM EDTA) plus the protease inhibitor cocktail (Roche). Protein samples (15-30 µg/lane) were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Memb-ranes were probed with antibodies directed against phosphotyrosine (1:1000, Millipore, Bedford, MA, USA), ErbB4 (1:1000, Santa Cruz Biotechnology). Alter-natively, immunoblots were probed with antibodies directed against excitatory and inhibitory neuronal markers GluA1 (1:500, Millipore), GluA2/3 (1:1000, Millipore), GluA4 (1:1000, Millipore), GluN1 (1:1000, Millipore), GluN2A (1:500, Millipore), GluN2B (1: 500, Millipore), GluN2C (1:500, Millipore), GluN2D (1:1000, Santa Cruz Biotechnology), GAD65/67 (1:1000, Millipore), parvalbumin (1:3000, Abcam, Cambridge UK), tyrosine hydroxylase (1:1000, Millipore) neuregulin-1 (1:300, RandD Systems, Minneapolis, MN), and β-actin (1:2000, Millipore). Immunoreactivity on membranes was detected by peroxidase-conjugated anti-immunoglobulin antibodies followed by chemiluminescence reaction (ECL kit, GE Healthcare) and film exposure. The intensity of an immunoreactive band, whose size matched the authentic molecular weight, was measured by an image processing software, GENETOOLS (Syngene, Cambridge, UK).

Kato T., Abe Y., Hirokawa S., Iwakura Y., Mizuno M., Namba H, & Nawa H. (2015). Neurobehavioral Differences Between Mice Receiving Distinct Neuregulin Variants as Neonates; Impact on Sensitivity to MK-801. Current Molecular Medicine, 15(3), 222-236.

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Western Blot Analysis of Brain Proteins

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All brain tissues were homogenized in RIPA buffer (Boster, Wuhan, China), with protease inhibitors and phosphatase inhibitors (Roche, Basel, Switzerland). The samples were centrifuged at 12,000 rpm for 15 min at 4°C and tissue debris was removed. Protein concentration was determined by BCA assay kit (Thermo Fisher Scientific, Waltham, United States). Protein samples (20∼40 μg per lane) were separated by SDS-PAGE and were then transferred into PVDF membranes (Millipore, Billerica, United States). Blocked for 60 min with 5% (w/v) non-fat milk in Tris-buffered solution-Tween 20, membranes were then incubated overnight at 4°C with PSD95 (1:500, Millipore, St. Charles, MO, United States), GAD65/67 (1:1000, Millipore, St. Charles, MO, United States), or β-actin (1:4000, Cell Signaling Technology, Boston, United States) primary antibody. Then they were incubated with HRP conjugated secondary anti-rabbit or anti-mouse IgG (Thermo Fisher Scientific, Waltham, United States) for 60 min at room temperature. The signals were detected using ECL kit (Millipore, St. Charles, MO, United States) and analyzed by Image J 1.46.

Ye X., Rong Z., Li Y., Wang X., Cheng B., Cheng Y., Luo H., Ti Y., Huang X., Liu Z., Zhang Y.W., Zheng W, & Zheng H. (2018). Protective Role of L-3-n-Butylphthalide in Cognitive Function and Dysthymic Disorders in Mouse With Chronic Epilepsy. Frontiers in Pharmacology, 9, 734.

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Immunohistochemical Labeling of Cortical Neurons

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Coronal sections 40 μm-thick containing the sensorimotor cortex were treated with 0.3% H₂O₂ in PBS to block the endogenous peroxidase activity, followed by treating with blocking solution to reduce the non-specific bindings. The sections were then incubated with primary antibodies against NeuN (1:500; Merck Millipore, Darmstadt, Germany), Foxp1 (1:1,000; Abcam, Cambridge, UK), Cux1 (1:1,000; Santa Cruz Biotechnology, Dallas, TX, USA), GAD65/67 (1:2,000; Sigma, St. Louis, MO, USA), Parvalbumin (Pvalb or PV, 1:3,000; Sigma) and Calretinin (Calb2 or CR, 1:3,000; Merck Millipore) at room temperature overnight. Next, sections were incubated with biotinylated secondary antibodies against mouse or rabbit IgG (1:500; The Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 2 h at room temperature followed by Vectastain [avidin-biotin complex (ABC) kit, Vector Laboratories, Burlingame, CA, USA] incubation for 1 h. Last, sections were processed with 2 mg/ml of 3,3′-Diaminobenzidine (DAB) with 0.01% H₂O₂ in PBS and mounted with a glycerol-based aqueous mounting medium.

Lee K.Y., Chang H.C., Seah C, & Lee L.J. (2019). Deprivation of Muscleblind-Like Proteins Causes Deficits in Cortical Neuron Distribution and Morphological Changes in Dendritic Spines and Postsynaptic Densities. Frontiers in Neuroanatomy, 13, 75.

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Immunohistochemical Analysis of Ki67 and GAD

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Blocking of endogenous peroxidase with 3% hydrogen
peroxide was performed on dewaxed and rehydrated slides
for 10 minutes. Heat induced epitope retrieval in citrate buffer
pH=6 (Abcam, UK) was used for 3 minutes (in microwave
oven) to recover the Ki67 epitope. Tris-EDTA buffer pH=9.0
(Abcam, UK) was used as the antigen retrieval procedure for
GAD65/67. Incubation with primary antibodies Ki67-clone
SP6 (Abcam, UK, dilution 1: 50) and GAD65/67 (Sigma,
USA, dilution 1: 1000), for two hours proceeded with the
application of biotinized secondary antibodies (Sigma, USA,
dilution 1: 400). Immunoreactivity was visualized using
extravidin peroxidase (Sigma, USA, dilution 1: 200) and 3,
3’-diaminobenzidine (SIGMAFAST™ DAB, Sigma, USA)
as chromogen. For each sample 5000 cells were counted and
the number of Ki67 and GAD65/67 positive cells per 1000
cells (‰) was determined. Images were taken with a Zeiss
Primo Star microscope.

Dzidziguri D., Modebadze I., Bakuradze E., Mosidze G, & Berulava M. (2017). Determination of The Properties of Rat Brain Thermostable Protein Complex which Inhibit Cell Proliferation. Cell Journal (Yakhteh), 19(4), 552-558.

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Visualization of TdTomato-positive Neurons

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Animals were perfused, fixed and sectioned as previously described (Mandela et al. 2012 ). β-galactosidase was visualized in coronal sections using X-Gal staining (Soriano 1999 (link)). TdTomato fluorescence was observed directly and nuclei were identified using the Hoechst stain. The identity of the TdTomato-positive neurons was further probed using immunocytochemistry for Vglut1 (1:3000, Chemicon, AB5905), GAD65/67 (1:2000, Sigma, G5163), Neuropeptide Y (JH4 [1:20,000]), and several products of POMC cleavage (Georgie [1:4000], 16K fragment; affinity-purified JH189 [1:500], γ₃MSH; JH44 [1:4000] and Kathy [1:4000], CLIP; JH93 [1:1000], N-ACTH) (Gaier et al. 2013 (link);Khachaturian et al. 1985 ;Ma et al. 2002 (link)).

Mandela P., Yan Y., LaRese T., Eipper B.A, & Mains R.E. (2014). Elimination of Kalrn Expression in POMC Cells Reduces Anxiety-Like Behavior and Contextual Fear Learning. Hormones and behavior, 66(2), 430-438.

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Immunofluorescence and Western Blot Antibodies

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The antibodies used in this study were NANOG (R&D Systems, AF1997, 1:500), OCT4 (STEMCELL Technologies, 60093, 1:1000), SOX1 (STEMCELL Technologies, 60095, 1:1000), SOX17 (R&D Systems, AF1924, 1:500), DESMIN (Invitrogen, PA5–16705, 1:500), MAP2 (Proteintech, 17490–1-AP, 1:500), TUJ1 (Neuromics, CH23005, 1:500), GFAP (STEMCELL Technologies, 60128, 1:500), TBR1 (Abcam, ab31940, 1:500), FOXG1 (Abcam, ab18259, 1:500), SYNAPSIN (EMD Millipore, 5747777, 1:500), VGLUT1 (Synaptic Systems, 135311, 1:500), GAD 65/67 (Sigma-Aldrich, G5163, 1:500), PGRN (R&D Systems, AF2420, 1:1000) (for immunofluorescence); PGRN (Sigma-Aldrich, HPA008763, 1:1000), CTSD (R&D Systems, AF1014, 1:1000), ACTIN (Novus Biologicals, NB600–532, 1:10,000) (for conventional western blot); V5 (Abcam, ab27671, 1:500), ACTIN (Novus Biologicals, NB600–532, 1:10,000) (for automated WES western blot) (Table S1).

Frew J., Baradaran-Heravi A., Balgi A.D., Wu X., Yan T.D., Arns S., Shidmoossavee F.S., Tan J., Jaquith J.B., Jansen-West K.R., Lynn F.C., Gao F.B., Petrucelli L., Feldman H.H., Mackenzie I.R., Roberge M, & Nygaard H.B. (2020). Premature termination codon readthrough upregulates progranulin expression and improves lysosomal function in preclinical models of GRN deficiency. Molecular Neurodegeneration, 15, 21.

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