Rabbit anti gad65 67

Manufactured by Merck Group

Rabbit anti-GAD65/67 is a primary antibody reagent that recognizes the glutamic acid decarboxylase (GAD) 65 and 67 isoforms. GAD is an enzyme involved in the synthesis of the neurotransmitter gamma-aminobutyric acid (GABA). This antibody can be used for the detection and analysis of GAD65 and GAD67 proteins in various research and diagnostic applications.

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5 protocols using rabbit anti gad65 67

Immunoblotting Protocol for Synaptic Proteins

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Protein extracts were resolved by SDS-PAGE, transferred onto nitrocellulose membrane (BioTraceNT, PALL), immunoblotted with the following primary antibodies: mouse anti-FMRP^{18 (link)} 1 µg/mL (DSHB, Clone 2F5-1); Rabbit anti-GluA1 C-terminal 1/1000 (Merk-Millipore #AB1504); Rabbit anti-GluA2 C-terminal 1/2000 (Synaptic System #182103); Rabbit anti-Homer1 1/1000 (Synaptic Systems #160003); Mouse anti-PSD95 1/10000 (NeuroMab #75-028 clone K28/43); Goat anti-GluN1 1/500 (Santa-Cruz #sc-1467); Mouse anti-Synapsin1a/b 1/500 (Santa-Cruz #sc-376623); Rabbit anti-GAD65/67 1/500 (Merk-Millipore #ABN904); Rabbit anti-CaMKII 1/500 (Santa-Cruz #sc-9035); Rabbit anti-Arc 1/1000 (Synaptic Systems #156003); Mouse anti-Gephyrin 1/1000 (Synaptic System #147111); Rabbit anti-vGluT1 1/5000 (Synaptic System #135303). Standard loading controls were included using a rabbit anti-GAPDH antibody 1/25000 (Sigma #G9545) or a rabbit anti-β3 Tubulin 1/25000 (Synaptic Systems #302302) as indicated. Proteins were revealed using the appropriate HRP-conjugated secondary antibodies (GE healthcare #NA931V and #NA934V). Proteins were then identified using Immobilon Western (Millipore) chemiluminescent solution and images acquired on a Fusion FX7 system (Vilber Lourmat). Full-size blots for cropped gels are shown in the Source data file.

Prieto M., Folci A., Poupon G., Schiavi S., Buzzelli V., Pronot M., François U., Pousinha P., Lattuada N., Abelanet S., Castagnola S., Chafai M., Khayachi A., Gwizdek C., Brau F., Deval E., Francolini M., Bardoni B., Humeau Y., Trezza V, & Martin S. (2021). Missense mutation of Fmr1 results in impaired AMPAR-mediated plasticity and socio-cognitive deficits in mice. Nature Communications, 12, 1557.

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Western Blotting of Synaptic Proteins

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Protein extracts were resolved by SDS-PAGE, transferred onto nitrocellulose membrane (BioTraceNT, PALL), immunoblotted with the following primary antibodies: mouse anti-FMRP 18 ; Rabbit anti-GluA1 C-terminal 1/1000 (Merk-Millipore); Rabbit anti-GluA2 C-terminal 1/2000 (Synaptic System); Rabbit anti-Homer1 1/1000 (Synaptic Systems); Mouse anti-PSD95 1/10000 (NeuroMab); Goat anti-GluN1 1/500 (Santa-Cruz); Mouse anti-Synapsin1a/b 1/500 (Santa-Cruz); Rabbit anti-GAD65/67 1/500 (Merk-Millipore); Rabbit anti-CaMKII 1/500 (Santa-Cruz); Rabbit anti-Arc 1/1000 (Synaptic Systems); Mouse anti-Gephyrin 1/1000 (Synaptic System); Rabbit anti-vGluT1 1/5000 (Synaptic System). Standard loading controls were included using a rabbit anti-GAPDH antibody 1/25000 (Sigma) or a rabbit anti-b3 Tubulin 1/25000 (Synaptic Systems) as indicated.
Proteins were revealed using the appropriate HRP-conjugated secondary antibodies (GE healthcare). Proteins were then identified using Immobilon Western (Millipore) chemiluminescent solution and images acquired on a Fusion FX7 system (Vilber Lourmat). Full-size blots for cropped gels are shown in Supplementary figure 7.

Prieto M., Folci A., Poupon G., Schiavi S., Buzzelli V., François U., Pousinha P., Lattuada N., Abélanet S., Pronot M., Castagnola S., Chafaï M., Khayachi A., Brau F., Deval E., Francolini M., Bardoni B., Humeau Y., Trezza V., & Martin S. (2020). Abnormal AMPAR-mediated synaptic plasticity, cognitive and autistic-like behaviors in a missense Fmr1 mutant mouse model of Fragile X syndrome.

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Comprehensive Immunofluorescence Profiling of Neural Cell Markers

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Cells were fixed with 4% paraformaldehyde (PFA) in PBS, permeabilized and blocked with 0.1% Triton-X100 plus and 5% normal goat serum in PBS. Then, they were incubated overnight at 4°C with the following primary antibodies: mouse anti-DLX2, 1:500 (Santa Cruz), rabbit anti-GABA, 1:1000 (Sigma), rabbit anti-GAD65/67, 1:250 (Millipore), mouse anti-GFAP, 1:200 (Millipore), mouse anti-MAP2, 1:200 (Chemicon), rabbit anti-MAP2, 1:1000 (Millipore), mouse anti-nestin, 1:200 (Chemicon), mouse anti-NeuN, 1:500 (Millipore), rabbit anti-neurofilament M, 1:200 (Millipore), mouse anti-NKX2.1, 1:1000 (Millipore), rabbit anti-OLIG2, 1:1000 (a gift from Dr. Charles Stiles, Harvard Medical School), mouse anti-PAX6, 1:250 (Millipore), mouse anti-PSD95, 1:500 (Millipore), rabbit anti-S100, 1:250 (Dako), mouse anti-SOX2, 1:500 (Millipore), rabbit anti-synaptophysin, 1:250 (Sigma), rabbit anti-TuJ1, 1:1000 (BioLegend) and mouse anti-vGAT, 1:200 (Synaptic Systems). After washing with PBS, cells were incubated with the secondary antibodies, Alexa Fluor® 555 anti-mouse IgG (Molecular Probes) and Alexa Fluor® 488 anti-mouse IgG (Molecular Probes). Cells were then counter-stained with 4,6-diamidino-2-phenylindole (DAPI) (Santa Cruz). The images were captured using a confocal laser-scanning microscope (LSM700; Zeiss) and digital inverted fluorescence microscope (DM5000B; Leica).

Park J., Lee N., Lee J., Choe E.K., Kim M.K., Lee J., Byun M.S., Chon M.W., Kim S.W., Lee C.J., Kim J.H., Kwon J.S, & Chang M.S. (2017). Small molecule-based lineage switch of human adipose-derived stem cells into neural stem cells and functional GABAergic neurons. Scientific Reports, 7, 10166.

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Quantitative Confocal Microscopy of Microglia-Neuron Interactions

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Confocal microscopy was performed as previously described8 (link). Briefly, free-floating coronal brain sections (30 μm) were pretreated with 1% Triton X-100 in PBS and blocked with 3% normal goat serum. Sections were incubated with primary antibodies (mouse-anti-Iba-1, 1:500, CCF Hybridoma Core; rabbit-anti-GAD 65/67, 1:300, Millipore; goat anti-pCREB, 1:250, Santa Cruz) overnight at 4 °C and then stained with appropriate Alexa-488 or -647 conjugated secondary antibodies (Vector Laboratories) for 2 h at room temperature (RT). Neurons were subsequently stained using Neuro Trace Nissl stain (1:300 for 30 min at RT, Life Technologies). Sections were rinsed, mounted with vectashield (Vector Laboratories) and examined on a Leica TCS confocal microscope (Leica Microsystems). The percentage of microglia in contact with neurons was calculated by dividing the number of microglia associated with neurons over the total number of microglia in a micrograph field. The percentage of neuronal circumference covered by inhibitory synapses was calculated by dividing the length of GAD 65/67 punctae around the neuronal soma by neuronal circumference.

Chen Z., Jalabi W., Hu W., Park H.J., Gale J.T., Kidd G.J., Bernatowicz R., Gossman Z.C., Chen J.T., Dutta R, & Trapp B.D. (2014). Microglial displacement of inhibitory synapses provides neuroprotection in the adult brain. Nature Communications, 5, 4486.

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Multimarker Fluorescence Immunocytochemistry

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Fluorescent immunocytochemistry followed standard protocols with primary antibodies for rabbit anti-FoxP1 (1:500; Abcam), mouse anti-FoxP1 (JC12) (1:500; Abcam), mouse anti-β-III-tubulin (1:1000; Sigma), mouse anti-Map2ab (1:500; Sigma), mouse anti-DARPP-32 (1:20,000; a gift from Prof H. Hemmings), mouse anti-GFAP (1:500; Abcam), rabbit anti-GAD65/67 (1:2000; Millipore), rabbit anti-met-enkephalin (1:15,000; Millipore), rat anti-CTIP2 (1:500, Abcam) and rat anti-BrdU (1:200, Oxford Bio). For double labelling, the two primary antibodies that had been raised in different species were added at the same time. Appropriate fluorescent-labelled secondary antibodies (Life technologies) were applied, followed by the nuclear stain Hoechst. Fluorescent staining was visualised using a Leica DRMBE microscope at 560 nm (red), 494 nm (green) and 346 nm (blue). Cell counts were undertaken at 40 × magnification using a counting grid. For unbiased sampling, 5 fields were chosen at random from which to take counts. Pseudocolour fluorescent images were obtained using Openlab 2.1 image analysis software and colour images were processed using Adobe Photoshop.

Precious S.V., Kelly C.M., Reddington A.E., Vinh N.N., Stickland R.C., Pekarik V., Scherf C., Jeyasingham R., Glasbey J., Holeiter M., Jones L., Taylor M.V, & Rosser A.E. (2016). FoxP1 marks medium spiny neurons from precursors to maturity and is required for their differentiation. Experimental Neurology, 282, 9-18.

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