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Synaptophysin

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Synaptophysin is a protein marker used in immunohistochemical analysis to detect synaptic vesicles in neuronal tissues. It is a widely used tool for the identification and characterization of neuronal and neuroendocrine cells.

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Lab products found in correlation

Psd95, by Abcam (8 mentions) Psd95, by Cell Signaling Technology (8 mentions) β actin, by Merck Group (8 mentions) Psd 95, by Merck Group (5 mentions) Glial fibrillary acidic protein (gfap), by Merck Group (5 mentions) Neuronal nuclei (neun), by Merck Group (5 mentions) Anti fading media, by Vector Laboratories (3 mentions) Glur1, by Merck Group (3 mentions) Bca kit, by Thermo Fisher Scientific (3 mentions) Fitc conjugated horse anti mouse igg secondary antibody, by Vector Laboratories (2 mentions) Confocal laser scanning microscope, by Leica (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions) Axioplan 2, by Zeiss (2 mentions) Vglut1, by Synaptic Systems (2 mentions) Protease inhibitor cocktail, by Roche (2 mentions) Glun2b, by Merck Group (2 mentions) Nestin, by Merck Group (2 mentions) Alexa fluor 488, by Thermo Fisher Scientific (2 mentions) Alexa 488, by Thermo Fisher Scientific (2 mentions) Alexa 555, by Thermo Fisher Scientific (2 mentions)

Spelling variants of this product

Anti-synaptophysin (42 citations) Mouse anti-Synaptophysin (22 citations) Rabbit anti-synaptophysin (6 citations) Mouse anti-synaptophysin antibody (4 citations) Rabbit-anti Neurofilament heavy or mouse anti-synaptophysin (2 citations) Mouse monoclonal anti-synaptophysin (2 citations) Anti-Synaptophysin (S5768) (2 citations)

80 protocols using synaptophysin

1

Isolation and Characterization of Synaptosomal Fractions

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Synaptosomes were prepared as described previously [6 (link), 22 (link)] with some modifications. Briefly, P56 mice (n = 4) were anaesthetized and perfused with sucrose buffer (0.32 M sucrose, 1 mM ethylenediaminetetraacetic acid, 5 mM dithiothreitol, pH 7.4). Whole brains were harvested and homogenized at 4 °C with a teflon-glass homogenizer using 12 strokes with 9:1 ratio of sucrose buffer supplemented with a protease cocktail inhibitor (Roche) to 1 g of tissue. Homogenate was centrifuged at 1000xg for 10 min at 4 °C. The resulting pellet containing mostly nuclei was removed and the supernatant was layered onto a discontinuous gradient consisting of 3, 10, 15 and 23 % (vol/vol) Percoll (GE Healthcare). Tubes were then centrifuged at 31,000xg for 8 min at 4 °C in a Sorvall WX Ultra90 (70.I TI rotor).
The contents of the resulting fractions have been characterized previously [6 (link), 31 (link)]. The resulting purified fractions were collected and protein was extracted in RIPA buffer for western blotting. Denatured protein samples (15 μg) were electrophoresed as described above. Membranes were probed with C9ORF72 antibody, along with synaptic markers: synaptophysin (1:5000, Millipore), PSD-95 (1:1000, Abcam), GAD67 (1:2500, Millipore); and GFAP (1:1000, NeuroMAB) as a marker of glia.
Atkinson R.A., Fernandez-Martos C.M., Atkin J.D., Vickers J.C, & King A.E. (2015). C9ORF72 expression and cellular localization over mouse development. Acta Neuropathologica Communications, 3, 59.
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2

Preparation and Use of Antibodies

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Rabbit anti-BEGAIN C17 and goat anti-PKCγ C14 antibodies were raised against C-terminal peptide SRKDSLTKAQLYGTLLN of mouse BEGAIN and mouse PKCγ (Yoshida et al., 2006 (link)), respectively. Commercially available antibodies against PSD-95 (Upstate Biotechnology, Lake Placid, NY), Hsp60 (Upstate Biotechnology), GluN2B (Millipore, Bedford, MA), synaptophysin (Millipore), glial fibrillary acidic protein (GFAP; Millipore), β-tubulin (Sigma, St. Louis, MO), and isothiocyanate-conjugated Bandeiraea simplicifolia isolectin B4 (IB4, Sigma) were also used.
Katano T., Fukuda M., Furue H., Yamazaki M., Abe M., Watanabe M., Nishida K., Yao I., Yamada A., Hata Y., Okumura N., Nakazawa T., Yamamoto T., Sakimura K., Takao T, & Ito S. (2016). Involvement of Brain-Enriched Guanylate Kinase-Associated Protein (BEGAIN) in Chronic Pain after Peripheral Nerve Injury. eNeuro, 3(5), ENEURO.0110-16.2016.
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3

Immunohistochemical Analysis of Neuronal Markers in AD Mouse Model

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Immunohistochemistry was performed on hAPP-J20 AD mouse brains and compared to WT littermate controls. Brains were fixed in freshly prepared 4% PFA and cut into 40 μm-thick vibratome sections. Immunolabeling was performed using mouse monoclonal antibodies against neuronal-specific proteins: microtubule associated protein 2 (MAP2, 1:100; Millipore) and synaptophysin (1:500; Millipore). After overnight incubation with primary antibody, sections were incubated with Texas red or FITC-conjugated horse anti-mouse IgG secondary antibody (1:75; Vector Laboratories) and mounted with anti-fading media (Vector). Immunosignals were analyzed by quantitative immunofluorescence using blind-coded sections, serially imaged on a laser-scanning confocal microscope, and quantified using NIH Image 1.43 software. At least three sections for each brain and four fields for each section were analyzed in each brain area.
Ghatak S., Dolatabadi N., Gao R., Wu Y., Scott H., Trudler D., Sultan A., Ambasudhan R., Nakamura T., Masliah E., Talantova M., Voytek B, & Lipton S.A. (2020). NitroSynapsin ameliorates hypersynchronous neural network activity in Alzheimer hiPSC models. Molecular psychiatry, 26(10), 5751-5765.
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4

Immunofluorescent Staining of Neural Markers

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Slides were baked for 60 min at 60 °C, deparaffinized with xylene for 5 min, rehydrated in ethanol (100%, 95%, 70%, and 50%). For antigen retrieval, slides were immersed in 10 mM sodium citrate and subjected to high heat and pressure for 20 min. After cooling, slides were rinsed with PBS for 10 min and blocked with 0.5% bovine albumin serum, 0.1% Triton X-100, and 2% fetal bovine serum in PBS for 30 min, and incubated overnight at 4 °C with a mix of primary antibodies MAP2 (Millipore, cat# AB5622, 1:200, Temecula, CA), synaptophysin (Millipore, cat# MAB368, 1:200, Temecula, CA), PSD-95 (abcam, cat# ab12093, 1:200, Cambridge, MA). After PBS rinses, slides were incubated with AlexaFluor conjugated secondary antibodies diluted in the blocking solution (donkey anti rabbit AF488, cat# A21206, 1:500; donkey anti mouse AF555, cat# A31570, 1:500; donkey anti goat AF647, cat# A21447, 1:500, Life Technologies, Carlsbad, CA) for 2 hours at room temperature. Slides were rinsed in PBS, counterstained with DAPI, lipofuscin autofluoresence was quenched with True Black (Biotum, cat# 23007, Hayward CA) according to manufacturer’s instructions, and coverslipped with Fluoromount G (Electron Microscopy Sciences, cat# 17984-25, Hatfield, PA).
Genç B., Jara J.H., Lagrimas A.K., Pytel P., Roos R.P., Mesulam M.M., Geula C., Bigio E.H, & Özdinler P.H. (2017). Apical dendrite degeneration, a novel cellular pathology for Betz cells in ALS. Scientific Reports, 7, 41765.
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5

Quantitative Neuropathological Analysis of Cyanide Exposure

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Neuropathological analysis was performed using methods previously published by our group (Talantova et al. 2013 (link); Meng et al. 2011 (link); Cho et al. 2011 (link)). To determine the effects of cyanide exposure and CA treatment, brains were fixed in 4% PFA, cut into 40 μm-thick vibratome sections and immunolabeled with mouse monoclonal antibodies against the neuronal-specific proteins, MAP2 (1:100; Millipore), NeuN (1:500; Millipore), and synaptophysin (1:500; Millipore). Primary antibodies were detected with horse anti-mouse FITC-conjugated antibody (1:75; Vector) and mounted under glass coverslips using anti-fading media (Vector). All sections were processed by standardized conditions. Staining was analyzed by quantitative confocal immunofluorescence using blind-coded sections, serially imaged on a laser-scanning confocal microscope, and quantified using NIH Image 1.43 software. A total of three sections for each brain and four fields for each section were analyzed in each brain area. For MAP2 and synaptophysin staining, results are expressed as percent area (% area) of the neuropil occupied by immunoreactive dendrites or terminals. NeuN-positive nuclei were counted stereologically using Stereo Investigator software (MicroBrightField, Wiliston, VT).
Zhang D., Lee B., Nutter A., Song P., Dolatabadi N., Parker J., Sanz-Blasco S., Newmeyer T., Ambasudhan R., McKercher S.R., Masliah E, & Lipton S.A. (2015). Protection from cyanide-induced brain injury by the Nrf2 transcriptional activator carnosic acid. Journal of neurochemistry, 133(6), 898-908.
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6

Immunostaining of Organotypic SGN Cultures

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After organotypic culture, SGN explants were incubated in ice-cold PBS at 4°C for 30 min and washed twice with PBS. In order to keep the architecture structure of SGNs, the coated matrigel was carefully removed except that the surrounded cultured tissue, followed by the fixation with 4% paraformaldehyde and permeabilization with 1% TritonX-100 in PBS (Sigma), samples were immersed in blocking solution (0.1% TritonX-100, 8% donkey serum, 1% bovine serum albumin, and 0.02% sodium azide in PBS) at room temperature for 1 h. Then, samples were incubated with different primary antibodies: β-tubulin (1 : 1000, Neuromics, USA), neurofilament (1 : 1000, Millipore, USA), and synaptophysin (1 : 1000, Millipore), diluted in blocking solution, respectively, at 4°C overnight. The next day, tissues were incubated with FITC-conjugated or TRITC-conjugated (1 : 1000, Invitrogen) secondary antibody along with DAPI (1 : 800, Sigma-Aldrich) or phalloidin (1 : 1000, Sigma-Aldrich) in 0.1% TritonX-100 and 1% BSA in PBS at room temperature for 1 h. Then, the coverslips were mounted and observed under a laser scanning confocal microscope (Leica, Germany).
Sun G., Liu W., Fan Z., Zhang D., Han Y., Xu L., Qi J., Zhang S., Gao B.T., Bai X., Li J., Chai R, & Wang H. (2016). The Three-Dimensional Culture System with Matrigel and Neurotrophic Factors Preserves the Structure and Function of Spiral Ganglion Neuron In Vitro. Neural Plasticity, 2016, 4280407.
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7

Antibody Analysis of Neurological Markers

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Antibodies include monoclonal antibody against NEP (clone Ab951) and PSD95 (Abcam), rabbit polyclonal antibody against NRG1 (clone SC-348) and anti-ErbB4 (Santa Cruz), anti-phospho-ErbB4 (Tyr1284) (Cell Signaling), mouse anti-β-actin antibody (Sigma), mouse monoclonal Aβ antibody 6E10 (Covance) and synaptophysin (Millipore).
Xu J., de Winter F., Farrokhi C., Rockenstein E., Mante M., Adame A., Cook J., Jin X., Masliah E, & Lee K.F. (2016). Neuregulin 1 improves cognitive deficits and neuropathology in an Alzheimer’s disease model. Scientific Reports, 6, 31692.
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8

Immunohistochemical Profiling of Retinal Cells

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Retinal sections were stained with the following antibodies with our published protocols (34 ,35 (link)) Cone-arrestin (rabbit polyclonal, 1:1,000; Millipore), PKCα (rabbit polyclonal, 1:5,000; Sigma), recoverin (rabbit polyclonal, 1:2,000; Millipore), calbindin D28k (rabbit polyclonal, 1:1,000; Swant), neurofilament-RT97 (mouse monoclonal, 1:1,000; Millipore), parvalbumin (rabbit polyclonal, 1:3,000; Swant), synaptophysin (mouse monoclonal, 1:2,000; Millipore), RPE65 (mouse monoclonal, 1:1,000; Millipore), type IV collagen (rabbit polyclonal, 1:1,000; Millipore), MAB1281 (mouse monoclonal, 1:300; Millipore), nestin (rabbit polyclonal, 1:2,000; Millipore), GFAP (rabbit polyclonal, 1:1,000; Sigma), S100β (mouse monoclonal, 1:250; Sigma), Ki67 (rabbit polyclonal, 1:500; Millipore), TuJ1 (mouse monoclonal, 1:1,000; Sigma), STEM121 (mouse monoclonal, 1:300; StemCells). Anti-mouse or rabbit secondary antibodies conjugated to Alexa Fluor-488 or Alexa Fluor-568 (Life Technologies) were used and counterstained with 49,69-diamidino-2-phenylindole (DAPI). Images were taken with a confocal microscope (Eclipse C1si; Nikon Instruments, Inc., Melville, NY).
Tsai Y., Lu B., Bakondi B., Girman S., Sahabian A., Sareen D., Svendsen C.N, & Wang S. (2015). Human iPSC-derived neural progenitors preserve vision in an AMD-like model. Stem cells (Dayton, Ohio), 33(8), 2537-2549.
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9

Protein Expression Analysis in Mouse Brain

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Mice hippocampi and cerebellums were collected and lysed with ice cold lysis buffer supplemented with 1% protease inhibitor cocktail (Pierce) and phosphatase inhibitor (100 uM NaF and 0.5 mM active Sodium orthovanadate). Protein concentrations were determined by Pierce BCA protein assay kit (Thermo Scientific), and the same amount of total protein was loaded to each lane. The quantification of target protein in each lane was normalized to the total amount of protein, which is determined by Ponceau S staining. Antibodies used included GluA1, p-GluA1, GluN1, PSD95 (all from Cell Signaling), and synaptophysin (Millipore).
Yu T., Taussig M.D., DiPatrizio N.V., Astarita G., Piomelli D., Bergman B.C., Dell’Acqua M.L., Eckel R.H, & Wang H. (2015). Deficiency of Lipoprotein Lipase in Neurons Decreases AMPA Receptor Phosphorylation and Leads to Neurobehavioral Abnormalities in Mice. PLoS ONE, 10(8), e0135113.
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10

Immunohistochemical Analysis of Neuronal Markers

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The procedures were performed as described in a previous study21 (link). The samples(frozen sections and cultured tissues) were incubated with different primary antibodies: SPAG621 (link), cleaved caspase-3 (1:400, Cell Signalling, USA, cytochrome C (1:400, Cell Signalling, USA) β-tubulin Ш (1: 1000, Neuromics, USA, a neuron-specific marker), neurofilament (1: 1000, Millipore, USA, a neuron-specific marker) and synaptophysin (1: 1000, Millipore) overnight. The next day, tissues were incubated with FITC conjugated or TRITC-conjugated (1: 1000, Invitrogen) secondary antibody along with DAPI (1: 800, Sigma-Aldrich) or phalloidin (1: 1000, Sigma Aldrich) for 1 hour. Then, the coverslips were mounted and observed under a laser scanning confocal microscope (Leica, Germany).
Li X., Xu L., Sun G., Wu X., Bai X., Li J., Strauss J.F., Zhang Z, & Wang H. (2017). Spag6 Mutant Mice Have Defects in Development and Function of Spiral Ganglion Neurons, Apoptosis, and Higher Sensitivity to Paclitaxel. Scientific Reports, 7, 8638.
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