Ab2723

Manufactured by Abcam

Sourced in United States, United Kingdom

Ab2723 is a recombinant monoclonal antibody targeting a specific protein. It is designed for use in various laboratory applications, including immunoassays and immunohistochemistry. The antibody is produced in a mammalian expression system and is purified using affinity chromatography.

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Lab products found in correlation

Ab8049, by Abcam (5 mentions) Ab31232, by Abcam (4 mentions) A11001, by Thermo Fisher Scientific (3 mentions) β actin, by Merck Group (2 mentions) Lsm 510 meta, by Zeiss (2 mentions) Lsm 780, by Zeiss (2 mentions) Vectashield, by Vector Laboratories (2 mentions) A11031, by Thermo Fisher Scientific (2 mentions) Normal goat serum (ngs), by Vector Laboratories (2 mentions) Mab3418, by Merck Group (2 mentions) D9542, by Merck Group (2 mentions) Paraformaldehyde (pfa), by Merck Group (2 mentions) Ab5905, by Merck Group (2 mentions) Ab5392, by Abcam (2 mentions) Lsm 710 confocal microscope, by Zeiss (2 mentions) Zen software, by Zeiss (2 mentions) Ab175678, by Abcam (2 mentions) Alexa fluor 488donkey anti, by Thermo Fisher Scientific (2 mentions) Cat 188 004, by Synaptic Systems (2 mentions) Cf 568 donkey anti mouse, by Biotium (2 mentions)

35 protocols using ab2723

Western Blot Analysis of Synaptic Proteins

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Samples from BLA tissues were harvested after various treatments according to our previously reported method (Yang et al. 2016 ). The western blot analysis determined the expression levels of GluA1 (1:1000; Abcam, ab31232), phosphorylated forms of GluA1 including phospho-GluA1-831 (1:1000; Abcam, ab5847) and phospho-GluA1-845 (1:1000; Abcam, ab5849), GluN2A (1:1000; Abcam, ab133265), GluN2B (1:1000; Millipore, Billerica, MA; MAB5780), PSD95 (1:1000; Abcam; ab2723), phosphorylated forms of JNK (1:1000; Cell Signaling Technology; Danvers, MA) and P38 (1:1000; Cell Signaling Technology), TNF-α (1:1000; Cell Signaling Technology), and p65 (1:1000; Cell Signaling Technology); β-actin (1:10,000; Sigma, St. Louis, MO) served as a loading control. The target protein signal was detected and digitized using ECL solution and ImageJ software (NIH, Bethesda, MD).

Sun T., Luo L., Tian Q.Q., Wang W.J., Liu Q.Q., Yang L., Zhang K., Zhang W., Zhao M.G, & Yang Q. (2020). Anxiolytic Effects of 8-O-Acetyl Shanzhiside Methylester on Acute and Chronic Anxiety via Inflammatory Response Inhibition and Excitatory/Inhibitory Transmission Imbalance. Neurotoxicity Research, 38(4), 979-991.

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Imaging Synaptic Proteins in Neurons

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Fixed primary hippocampal neurons were treated with 0.1 % of the reducing agent NaBH₄ (prepared immediately before use) for 7 min at RT prior to permeabilization in 0.4 % CHAPSO. Blocking steps were done essentially as described above for confocal microscopy. In the first antibody incubation step, a combination of either of mouse anti-synaptophysin IgG diluted 1:1000 (SP15, Enzo Life Sciences), mouse anti-PSD95 IgG diluted 1:100 (Ab2723, Abcam) or mouse anti-NMDAR2B (610416, BD Transduction Laboratories) with 10 μM (Z-LL)₂ ketone and 200 nM GTB ± 10 μM L685,458 were used. The second incubation buffer, used after washing and UV illumination, contained Cy3/Alexa647-conjugated streptavidin (~30 nM) and Alexa405/Alexa647-conjugated anti-mouse IgG diluted 1:100 in 3–5 % NGS. Washing (conducted as in sample preparation for confocal microscopy) was followed by postfixation in 3 % formalin/0.1 % glutaraldehyde for 10 min at RT, followed by washing 3 × 5 min with DPBS and 1 × 5 min with water. Negative controls lacking either of the primary antibodies or GTB were also prepared.

Schedin-Weiss S., Caesar I., Winblad B., Blom H, & Tjernberg L.O. (2016). Super-resolution microscopy reveals γ-secretase at both sides of the neuronal synapse. Acta Neuropathologica Communications, 4, 29.

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Immunoprecipitation and Western Blot Analysis of Hippocampal Proteins

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The hippocampal tissue from 8-week mice was lysed in lysis buffer with 1 mM PMSF and centrifugated at 14,000 g for 15 min. For immunoprecipitation, the supernatant lysate was incubated with rabbit anti-SHANK3 (64555S, Cell Signaling Technology, USA; 1:50) or IgG antibody (2729S, Cell Signaling Technology, USA; 1:1,000) at 4°C overnight. Then the mixture was incubated with Protein A/G Plus-Agarose (SC-2003, Santa Cruz, USA) for 3 hours at 4°C. After centrifugation at 1,000 g for 5 min at 4°C, the immunoprecipitants were washed four times with lysis buffer and then boiled in protein loading buffer for 5 min. Western blotting was performed to detect the proteins using the following primary antibodies: rabbit anti-SHANK3 (64555S, Cell Signaling Technology, USA; 1:1,000), rabbit anti-GluN1 (5704S, Cell Signaling Technology, USA; 1:1,000), rabbit anti-GluN2B (14544S, Cell Signaling Technology, USA; 1:1,000), rabbit anti-GABRB1 (20183-1-AP, Proteintech, USA; 1:800), mouse anti-GAPDH (60004-1-Ig, Proteintech, USA; 1:10,000), mouse anti-PSD95 (ab2723, Abcam, UK; 1:1,200), rabbit anti-Synaptophysin (ab52636, Abcam, UK; 1:4,000).

Wang J., Gao Y., Xiao L., Lin Y., Huang L., Chen J., Liang G., Li W., Yi W., Lao J., Zhang B., Gao T.M., Zhong M, & Yang X. (2023). Increased NMDARs in neurons and glutamine synthetase in astrocytes underlying autistic-like behaviors of Gabrb1−/− mice. iScience, 26(8), 107476.

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Assessing Synaptic and Glial Changes in Kindling Model

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During every Racine stage (1–5) in the kindling group or on the 10th day of amygdala kindling in drug/siRNA treated groups, four rats out of each group were deeply anesthetized and perfused intracardially with 4% paraformaldehyde in PBS. Coronal slices, 10 μm thick, were prepared by using a cryostat (CM3050s, Leica, Germany). In every group, immunofluorescence staining for post synaptic density protein 95 (PSD-95, 1:200, Abcam, ab2723) and double-immunofluorescence staining for glial fibrillary acidic protein (GFAP, 1:100, Beijing Zhongshan, ZA-0117)/TSP-1 (1:100, Abcam, ab1823) were performed. Sequentially, the sections were incubated with secondary antibodies (fluorescein isothiocyanate (FITC)-conjugated, 1:200, EMD Millipore; cyanine-3 (Cy3)-conjugated, 1:200, Beyotime Institute of Biotechnology), after washing it thrice with 0.01M PBS, the sections were coverslipped and observed under a fluorescence microscope (CX41, Olympus, Japan). In addition, the optical density of IR was quantified with ImageJ 1.37 software (NIH, Bethesda, MD, USA). For additional analysis, three fields (80 μm × 60 μm/field) were selected randomly in every 200× microscope view, and PSD-95-positive puncta in the fields were counted and averaged.

Sun H., Ma L., Zhang Y., Pan X., Wang C., Zhang J., Zhang X., Sun H., Wang Q, & Zhu W. (2018). A Purinergic P2 Receptor Family-Mediated Increase in Thrombospondin-1 Bolsters Synaptic Density and Epileptic Seizure Activity in the Amygdala-Kindling Rat Model. Frontiers in Cellular Neuroscience, 12, 302.

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Characterization of Hippocampal Proteins

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Hippocampal tissues and cultured neurons were lysed in buffer containing 25 mM HEPES at pH7.9, 150 mM NaCl, 1 mM PMSF, 20 mM NaF, 1 mM DTT, 0.1% NP40, and proteinase inhibitor cocktails (Roche). Protein concentrations were determined by Folin phenol method with bovine serum albumin as standard. Twenty micrograms of the protein was separated on 8–12% SDS-PAGE gels (Bio-Rad) and transferred to PVDF membranes (Millipore). The membranes were blocked in 5% BSA in TBS-T with 0.05% Tween-20 and incubated with primary antibodies at 4°C overnight. Dilutions of primary antibodies were 1:1000 for UTX (E409, Millipore), H3K27me3 (1:1,000, 07449, Millipore), PSD95 (ab2723, Abcam), Synapsin (ab8049, Abcam), and 1:10,000 for β-actin antibody (Sigma). As for the secondary antibodies, we used HRP-linked goat anti-mouse or HRP-Linked goat anti-rabbit at 1:500. Enhanced chemoluminescence (ECL, Pierce) was used for detection. Quantification of the blots was determined with Quantity One Ver.4.4.0 (BioRad, USA).

Tang G.B., Zeng Y.Q., Liu P.P., Mi T.W., Zhang S.F., Dai S.K., Tang Q.Y., Yang L., Xu Y.J., Yan H.L., Du H.Z., Teng Z.Q., Zhou F.Q, & Liu C.M. (2017). The Histone H3K27 Demethylase UTX Regulates Synaptic Plasticity and Cognitive Behaviors in Mice. Frontiers in Molecular Neuroscience, 10, 267.

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Immunostaining of Neuronal Markers

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Cells were fixed with 4% paraformaldehyde/sucrose in PBS for 5min and then permeabilized with 0.1% Triton X-100 in PBS for 10min at room temperature. Cells were blocked with 10% FBS in PBS for at least 30min at room temperature, and incubated with the primary Ab overnight with 3% FBS in PBS. Primary antibodies used were as follows: rabbit anti-βIII-tubulin (T2200, 1:1000, Sigma), rabbit anti-synapsin 1,2 (106002, 1:200, Synaptic systems), mouse anti-PSD95 (ab2723, 1:200, Abcam). Samples were then washed and incubated with secondary conjugated Ab (1:500; anti-rabbit AF594, A11072; anti-rabbit AF633, A31573; anti-mouse AF594, A11005; anti-mouse AF633, A21050; all from Invitrogen and anti-mouse FITC, F2012, from Sigma) for 1h at room temperature. After washing, samples were mounted in VectaShield (Vector Laboratories). Cells were examined on LSM 510 Meta or LSM 780 (Zeiss) confocal microscopes.

Ferron L., Nieto-Rostro M., Cassidy J.S, & Dolphin A.C. (2014). Fragile X mental retardation protein controls synaptic vesicle exocytosis by modulating N-type calcium channel density. Nature Communications, 5, 3628.

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Quantitative Analysis of Neuronal Markers in Mouse Brain

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Mice (n=3-5/group) were deeply anesthetized with isoflurane (3–5%) in oxygen (0.8 L/min) and fixed transcardially with paraformaldehyde (4%, w/v). Brain were post-fixed overnight in paraformaldehyde (4%, w/v), embedded in paraffin, and parasagittal sections (10μm) prepared 0.8-2mm from the midline (HistoWiz, Brooklyn, NY). Sections were stained with antibodies against MAP2 (Abcam, catalog #ab32454; antibody registry ID: AB 776174; rabbit polyclonal 1:500), NeuN (Abcam, catalog #ab104225; antibody registry ID: 10711153; rabbit polyclonal 1:500), synaptophysin (LifeSpanBioSciences, catalog #LS-C203763; antibody registry ID: AB 2864290; rabbit polyclonal, 1:500), and PSD-95 (Abcam, catalog #ab2723; antibody registry ID: 303248; mouse monoclonal, 1:500) with the appropriate fluorescent secondary antibodies. Nuclei were stained with 4′,6-diamidino-2-phenylindole. The specificity of each antibody binding was tested by staining adjacent regions with only primary or secondary antibodies. Sections were imaged using an Olympus Fluoview FV100 laser scanning confocal microscope using a 60x oil immersion objective and Leica Application Suite 1.8.2. Image J software measured MAP2 immunoreactivity in CA3 and CA1. Changes in MAP2 protein expression were examined separately in apical and basal dendritic trees.

Whitney K., Nikulina E., Rahman S.N., Alexis A, & Bergold P.J. (2021). Delayed dosing of Minocycline plus N-acetylcysteine reduces neurodegeneration in distal brain regions and restores spatial memory after experimental traumatic brain injury. Experimental neurology, 345, 113816.

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Hippocampal Protein Expression Analysis

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Hippocampus was homogenized by motor-driven pestle in cold RIPA buffer (20–188, Millipore, Temecula, CA, USA) and centrifuged 5 min/20 000 g. Supernate protein (20 μg) was electrophoresed on 10% SDS polyacrylamide gels and transferred to polyvinylidene fluoride membranes. The polyvinylidene fluoride membranes were blocked with 5% BSA for 1 h and probed with primary antibodies overnight at 4 °C: anti-GluR1 (glutamate receptor subunit 1; 1:3000, AB31232, rabbit; Abcam, Cambridge, MA, USA), anti-GluR2 (glutamate receptor subunit 2; 1:2000, AB1768, rabbit; Millipore), anti-NR2A (NMDA receptor, subunit 2A, 1:1000, 07-632, rabbit; Millipore), anti-NR2B (NMDA receptor, subunit 2B, 1:1000, 06-600, rabbit; Millipore) anti-PSD95 (1:1000, AB2723, mouse; Abcam), anti-synaptophysin (1:5000, MAB368, mouse; Stressgene; Enzo, Plymouth Meeting, PA, USA), and anti-NueN (loading control; 1:1000, MAB377, mouse, Millipore). After washing, membranes were probed with secondary antibodies conjugated with IRDye 680 (92632210, rabbit, LI-COR Biosciences, Lincoln, NE, USA) and IRDye 800 (92632210, mouse, LI-COR). Signal was detected by infrared imaging (Odyssey, LI-COR).

Cacciottolo M., Wang X., Driscoll I., Woodward N., Saffari A., Reyes J., Serre M.L., Vizuete W., Sioutas C., Morgan T.E., Gatz M., Chui H.C., Shumaker S.A., Resnick S.M., Espeland M.A., Finch C.E, & Chen J.C. (2017). Particulate air pollutants, APOE alleles and their contributions to cognitive impairment in older women and to amyloidogenesis in experimental models. Translational Psychiatry, 7(1), e1022-.

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Immunocytochemical Staining of Neuronal Markers

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ICC staining occurred as described previously^{13 (link)}. In brief, cells were first preblocked using DPBS containing 5% normal goat serum (Vector Laboratories) and 0.1% Triton X-100 for 30 minutes, followed by overnight incubation with primary antibodies: mouse microtubule-associated protein 2 (MAP2, 1:400; MAB3418; Millipore), mouse postsynaptic density protein 95 (PSD-95; 1:500; ab2723; Abcam), or mouse synaptophysin (1:300; ab8049; Abcam). After washing with DPBS, cells were incubated with secondary antibodies that included goat anti-mouse Alexa Fluor 568 (1:500; A11031; Invitrogen) or goat anti-mouse Alexa Fluor 488 (1:500; A11001; Invitrogen), followed by counterstaining with DAPI (D9542; Sigma-Aldrich). Coverslips were then mounted on slides with Mowiol mounting medium.

Cheng Y., Wang Z.M., Tan W., Wang X., Li Y., Bai B., Li Y., Zhang S.F., Yan H.L., Chen Z.L., Liu C.M., Mi T.W., Xia S., Zhou Z., Liu A., Tang G.B., Liu C., Dai Z.J., Wang Y.Y., Wang H., Wang X., Kang Y., Lin L., Chen Z., Xie N., Sun Q., Xie W., Peng J., Chen D., Teng Z.Q, & Jin P. (2018). Partial loss of psychiatric risk gene miR-137 in mice causes repetitive behavior and impairs sociability and learning via increased Pde10a. Nature neuroscience, 21(12), 1689-1703.

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Immunocytochemistry of Hippocampal Neurons

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For immunocytochemistry of the hippocampal neuronal cultures and mixed culture assays, cells were permeabilized and blocked with 3% fetal bovine serum, 3% BSA, and 0.1% Triton X-100 in PBS for 15 minutes at room temperature. Primary antibodies were applied overnight at 4°C, and subtype-specific Alexa Fluor fluorescent secondary antibodies were added for 40 minutes at room temperature. Coverslips were mounted with ProLong Gold antifade reagent (Invitrogen; P36934). Primary antibodies were anti-PSD95 (Abcam; catalog ab2723), anti-MAP2 (Abcam; catalog ab92434), anti-bassoon (Enzo, ADI-VAM-PS003-D clone, catalog SAP7F407), anti-GluR1 (Invitrogen, catalog PA1-46151), and anti-p75^NTR (R&D Systems; catalog AF1157). Secondary antibodies were Alexa Fluor antibodies (Life Technologies), except for the 405 nm fluorescent DyLight (Jackson ImmunoResearch Laboratories; catalog 102649-302). For visualization of actin cytoskeleton, Alexa Fluor 546 and Alexa Fluor 647 phalloidin (Life Technologies, Thermo Fisher Scientific; A22283 and A30107) were used.

Morano N.C., Smith R.S., Danelon V., Schreiner R., Patel U., Herrera N.G., Smith C., Olson S.M., Laerke M.K., Celikgil A., Garforth S.J., Garrett-Thomson S.C., Lee F.S., Hempstead B.L, & Almo S.C. (2022). Human immunomodulatory ligand B7-1 mediates synaptic remodeling via the p75 neurotrophin receptor. The Journal of Clinical Investigation, 132(22), e157002.

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