DPA or DPD cells were incubated at a density of 10 × 106 cells/ml with or without 2 μg/ml GAD65 (Abcam) in PBS + 1% BSA on ice for 1 h to load GAD65 onto BCR while BCR internalization is inhibited. GAD65-loaded or non-loaded cells were washed with PBS + 1% BSA, pelleted by centrifugation at 400 x g, 4 °C for 5 min, resuspended at a density of 2 × 106 cells/ml in complete IMDM GlutaMAX medium containing 5μg/ml GAD65 or no antigen, and then incubated at 37 °C for an indicated time length. At 0, 20 min, 1 h, and 2 h, 0.6 million cells were collected by centrifugation at 400 x g, 4 °C for 5 min and used in three different assays: 1) 0.2 million cells were used for iFACS analysis (see below). 2) 0.2 million cells were incubated at a density of 10 × 106 cells/ml with 10 μg/ml Alexa Fluor 488-conjugated goat anti-human IgG in PBS + 1% BSA on ice for 30 min to stain surface BCR. 3) 0.2 million cells were incubated at a density of 10 × 106 cells/ml with 10 μg/ml anti-GAD65 antibody on ice for 30 min to stain surface GAD65, followed by two washes with PBS + 1% BSA and the secondary staining with 10 μg/ml Alexa Fluor 488-conjugated goat anti-mouse IgG(H + L) in PBS + 1% BSA on ice for 30 min. Antibody-labeled cells in 2) and 3) were analyzed on a BD FACSCalibur flow cytometer.
Gad65
Manufactured by Abcam
Sourced in United Kingdom, United States
GAD65 is a protein that functions as an enzyme involved in the production of the neurotransmitter gamma-aminobutyric acid (GABA) in the brain and other tissues. It plays a crucial role in the regulation of GABA levels, which is important for neurological function and signaling.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Abcam (2 mentions)
Synaptophysin, by Santa Cruz Biotechnology (1 mentions)
App c ter, by Merck Group (1 mentions)
Total tau, by Santa Cruz Biotechnology (1 mentions)
Psd 95, by Thermo Fisher Scientific (1 mentions)
Ecl anti rabbit horseradish peroxidase linked, by GE Healthcare (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Neuronal nuclei (neun), by Merck Group (1 mentions)
App 6e10, by Fortrea (1 mentions)
Actin, by Abcam (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Ecl system, by Thermo Fisher Scientific (1 mentions)
α tubulin, by Merck Group (1 mentions)
Anti psd95, by Thermo Fisher Scientific (1 mentions)
Gephyrin, by Synaptic Systems (1 mentions)
Glur1, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Glutamine synthetase, by BD (1 mentions)
Isolectin b4, by Vector Laboratories (1 mentions)
Control igg, by BioLegend (1 mentions)
9 protocols using gad65
1
BCR Internalization and GAD65 Binding Assay
2
T Cell Proliferation Assay with CFSE
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T lymphocytes were stained with carboxyfluorescein succinimidyl ester (CFSE). The concentration of CFSE fluorescent dye is reduced by cell division. During cell division, CFSE is distributed among daughter cells. Hence, proliferation is assessed by dilution of the CFSE dye (34 (link)). Briefly, 1x106 lymphocytes were incubated with CFSE (10mM; Abcam, UK) for 10 minutes in a 5% CO2 incubator at 37°c and 95% relative humidity. The reaction was stopped with RPMI supplemented with 10%PL. T Lymphocytes were co-cultured with GAD65 pulsed mDCs (mDCs: T cell ratio, 1:10) in 6-well plates with RPMI 1640 supplemented with 10% PL, 100u/ml penicillin/streptomycin, 2Mm/ml L-Glutamine, and pulsed at concentration of 1x106 cells/ml with 10µg/ml GAD65 (Abcam, UK) for 4 days (referred to as activated T cells). After that, activated T cells (CD4+ and CD8+) were tested using flow cytometry. In a parallel experiment, T lymphocytes were cultured alone under the same conditions without activation (referred to as inactivated T cells).
3
Western Blot Analysis of Neurological Proteins
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Equal amounts of protein (30 μg) were separated by electrophoresis in NuPAGE Bis-Tris Gels (Life Technologies) and transferred to nitrocellulose membranes. The membranes were hybridized with various primary antibodies (APP 6E10, 1/500, Covance; PS1, 1/1000, Millipore; APP C-ter, 1/500, Millipore; Actin, 1/2000, Abcam; GAPDH, 1/1000 Abcam; Total Tau, 1/1000, Santa Cruz; PSD-95, Invitrogen, 1/2000; Synaptophysin, Santa Cruz, 1/200; GAD65, Abcam, 1/2000; GLT-1, 1/1000, Frontier Science; GLAST, 1/1000, Frontier Science; NeuN, Millipore, 1/1000). Various secondary antibodies was also used (ECL Anti-rabbit Horseradish Peroxidase linked, 1/2000, GE Healthcare; ECL Anti-mouse Horseradish Peroxidase linked, 1/2000, GE Healthcare; ECL Anti-rat Horseradish Peroxidase linked, 1/2000, GE Healthcare).
4
Hippocampus and Frontal Cortex Protein Profiling
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Proteins of mice hippocampus and frontal cortex was prepared after 3 injections of SCE, AA, SCE and AA mixture (10 mg/kg) using an RIPA buffer consisting of 50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate and 1% Nonidet P-40. A 10% phosphatase inhibitor and 10% protease inhibitor cocktail (Roche, Basel, Switzerland) was added before using the RIPA buffer. Next, 12 μg of protein was mixed with a 5X sample buffer and loaded on SDS-PAGE gel. The Protein sample was run on gel by electrophoresis and transferred by 200 mA to polyvinylidene fluoride (PVDF) membrane (Millipore, MA, USA). The membrane was blocked by 5% skim milk and incubated with primary antibodies including anti-PSD95 (Thermo Scientific, MA, USA), GluR1 (Abcam, Cambridge, UK), GAD65 (Abcam, Cambridge, UK), Gephyrin (Synaptic Systems, Goettingen, Germany), and α-Tubulin (Sigma-Aldrich, MO, USA) antibodies at 4 °C overnight. The membrane was incubated with a secondary anti-mouse or rabbit IgG horseradish peroxidase antibody (HRP, Pierce Biotechnology, MA, USA), which corresponds to the primary antibody host, for 1 h at room temperature and each protein band was visualized by the ECL system (Thermo Scientific, MA, USA). For ECL detection, we used medical X-ray film blue (AGFA CP-BU NEW, Belgium), developer solution, and fixer solution for the ECL detection.
5
Detecting Cell Signaling Pathways in CNV Model
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Antibodies used for this study include rabbit antibodies against VEGFR2 (Cell Signaling Technology, Danvers, MA, USA) and GAD65 (Abcam, Cambridge, MA, USA), goat antibody against Centaurin A (Santa Cruz Biotechnology, Dallas, TX, USA), mouse antibodies against rhodopsin (Millipore, Burlington, MA, USA) and glutamine synthetase (BD Bioscience, Franklin Lakes, NJ, USA). To neutralize VEGF in the mouse laser-induced CNV model, a mouse-specific antibody against VEGF (512807; BioLegend, San Diego, CA, USA) and the IgG control (400515; BioLegend) was purchased from the same company. Aflibercept was obtained from the eye clinic adjacent to the Department of Ophthalmology at UIC. Other reagents used were isolectin B4 (Vector Laboratories, Burlingame, CA, USA), Alexa 594 anti-rabbit antibody, Alexa 488 anti-mouse antibody, Alexa 488-streptavidin (Invitrogen, Carlsbad, CA, USA), and HRP-conjugated secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA, USA).
6
Molecular Markers for Neurodegenerative Diseases
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The following reagents and antibodies were used for this study: HIV‐1 Tat 101 (Immunodiaognistics, Woburn, MA), mutant Tat (ab83352, Abcam, MA, USA), HIF‐1α siRNA (sc‐35561, Santa Cruz Biotechnology, TX, USA), GW4869 (D1692, Sigma‐Aldrich, MO, USA), GFP‐plasmid (13031, Adgene, MA, USA), antibodies‐ AβmOC64 (ab201060, Abcam, MA, USA), APP (ab15272, Abcam, MA, USA), TSG101 (ab125011, Abcam, MA, USA), Ago‐2 (ab186733, Abcam, MA, USA), Alix (ab275377, Abcam, MA, USA), Calnexin (ab133615, Abcam, MA, USA), Flotillin (ab133497, Abcam, MA, USA), CD63 (ab216130, Abcam, MA, USA), Drebrin (ab178408, Abcam, MA, USA), GAD65 (ab239372, Abcam, MA, USA), Gephyrin (ab181382, Abcam, MA, USA), GFAP (G3893, Sigma‐Aldrich, MO, USA), Goat anti‐mouse (sc‐2005, Santa Cruz Biotechnology, TX, USA), Goat anti‐rabbit (sc‐2004, Santa Cruz Biotechnology, TX, USA), HIF‐1α (NB100‐449, Novus Biological Company, CO, USA), Iba1 (ab17884, Abcam, MA, USA) IL‐6 (ab179570, Abcam, MA, USA), Tau (ab9778, Abcam, MA, USA), p‐tau 214 (ab170892, Abcam, MA, USA), p‐Tau 622 (ab76128, Abcam, MA, USA), Synaptophysin (ab8049 Abcam, MA, USA), vGLUT1 (AB5905, Millipore, MA, USA), NeuN (ab134014, Abcam, MA, USA), MAP 2 (ab5392, Abcam, MA, USA), β‐Actin (A5316, Sigma‐Aldrich, MO, USA) were purchased from commercial vendors.
7
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).
8
Nanopore Detection of GAD65 and GADAb
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GAD65 was purchased from Abcam (Cambridge, UK) and GADAb (both monoclonal and polyclonal) were purchased from Sigma-Aldrich (Shanghai, China). Tris-HCl (pH = 8.0) was produced by Solarbio (Beijing, China). Both KCl and ethanol were purchased from Sinopharm (Shanghai, China). Electrolyte for translocation through nanopore was 1 M KCl solution contain 10 mM Tris-HCl (pH = 8.0). All solutions were prepared with Milli-Q water (18 MΩ·cm resistivity) from a Millipore system (Merck, Darmstadt, Germany).
9
Hippocampus Protein Expression Analysis
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The hippocampus of mice was homogenized, sectioned and analyzed by Western blotting. Totally thirty micrograms of protein were separated from each sample by 10% SDS-PAGE electrophoresis. After transferred to polyvinylidene uoride membrane (GE Healthcare, Little Chalfont, Buckinghamshire, UK), the proteins were incubated with respective primary antibodies against glutamate decarboxylase 2 (GAD65) (cat: ab26113), EGR-1 (cat: ab194357), BDNF (cat: ab88901), TrkB (cat: ab101696) and GAPDH (cat: ab181602) provided by Abcam (Cambridge, MA, USA). Sequentially, the proteins were further incubated with secondary antibodies as described previously [16] . The protein bands were visualized by ECL reagent kit (cat: RPN2232, GE Healthcare) and quanti ed with Gel-Pro analyzer software (Media Cybernetics, L.P., MD, Rockville, USA).
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