Mouse anti synaptophysin

Manufactured by Merck Group

Sourced in United States

Mouse anti-Synaptophysin is a laboratory reagent used to detect the synaptic vesicle protein synaptophysin in various biological samples. It is a primary antibody that specifically binds to the synaptophysin protein, allowing for its identification and localization in cells and tissues.

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Close spelling variants of this product

Mouse monoclonal anti-synaptophysin Mouse anti-synaptophysin antibody Rabbit-anti Neurofilament heavy or mouse anti-synaptophysin Anti-synaptophysin Synaptophysin

22 protocols using mouse anti synaptophysin

LRRK2 and synapse protein detection

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The primary antibodies used in this study are rabbit anti-LRRK2 1:500 MJFF C41-2, rabbit anti-LRRK2 P-Ser-935 UDD2 10(12) (Abcam, Cambridge, UK), mouse anti-actin 1:1000, mouse anti-FLAG 1:1000, mouse anti myc 1:1000, mouse anti-synaptophysin 1:1000 (Sigma-Aldrich St. Louis, MO, USA).

Stanic J., Mellone M., Cirnaru M.D., Perez-Carrion M., Zianni E., Di Luca M., Gardoni F, & Piccoli G. (2016). LRRK2 phosphorylation level correlates with abnormal motor behaviour in an experimental model of levodopa-induced dyskinesias. Molecular Brain, 9, 53.

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Western Blot Analysis of Cerebral Cortex

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Fresh frozen cerebral cortex was homogenized in RIPA buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 10 mM EDTA, 2 mM EGTA, 50 mM NaF, 0.5% SDS, 1% NP-40) containing a protease inhibitor cocktail (Roche Applied Science). Protein concentrations were determined by BCA (Thermo Scientific). Proteins were separated on an SDS-PAGE gel, and transferred to a nitrocellulose membrane (Bio-Rad). Membranes were blocked in TBS (100 mM Tris-Cl, pH 7.5, 150 mM NaCl) containing 0.1% Tween-20, and 5% nonfat milk solution for 1 h, then incubated with one of the following primary antibodies: mouse-anti-PSD95 (1:1000; EMD Biosciences), mouse-anti-synaptophysin (1:500; Sigma), or mouse anti-βIII-Tubulin (1:2000; Sigma) overnight at 4 °C on a rotary shaker. Membranes were washed three times with 0.1% TBS prior to addition of an anti-mouse IgG horse radish peroxidase secondary antibody (1:2000; Cell Signaling Technology). Proteins were visualized by chemiluminescence (Millipore) on a QBOX imaging system (Syngene). Densitometry analysis was performed in ImageJ (NIH) using the gel analyzer plug-in.

Dickens A.M., Yoo S.W., Chin A.C., Xu J., Johnson T.P., Trout A.L., Hauser K.F, & Haughey N.J. (2017). Chronic low-level expression of HIV-1 Tat promotes a neurodegenerative phenotype with aging. Scientific Reports, 7, 7748.

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Endocytosis Assay for SV Recycling

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The endocytosis assay to monitor SV recycling was performed using rabbit polyclonal antibodies directed against the intravesicular domain of synaptotagmin1 (Synaptic System), applied for 5 min if not indicated otherwise at RT on the cultures, as described previously (Matteoli et al., 1992 (link)). Incubations with the antibody (1:400) were performed in Tyrode solution containing 124 mM NaCl, 5 mM KCl, 2 mM MgCl₂, 30 mM glucose, 25 mM HEPES, pH 7.4 and 2 mM CaCl₂. After fixation and permeabilization, a synaptophysin counter staining with mouse anti synaptophysin, 1:400 (Sigma-Aldrich) visualized the totality of SV. Acquired images were processed and quantitatively analyzed with ImageJ software as previously described (Verderio et al., 1999 (link)). Briefly, GFP positive processes were manually tracked and the number of synaptotagmin and synaptophysin positive clusters and synaptophysin positive clusters present in the region of interest were automatically counted.

Cirnaru M.D., Marte A., Belluzzi E., Russo I., Gabrielli M., Longo F., Arcuri L., Murru L., Bubacco L., Matteoli M., Fedele E., Sala C., Passafaro M., Morari M., Greggio E., Onofri F, & Piccoli G. (2014). LRRK2 kinase activity regulates synaptic vesicle trafficking and neurotransmitter release through modulation of LRRK2 macro-molecular complex. Frontiers in Molecular Neuroscience, 7, 49.

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Immunocytochemical Characterization of Neural Cells

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The control plate cells were fixed after 12 days in adherent culture, and immunocytochemical staining was performed as previously described (Lappalainen et al., 2010 (link)). Primary antibodies, rabbit polyclonal anti-Microtubule-Associated Protein 2 (MAP2) (1:400; Millipore), mouse anti-beta-III Tubulin (β-tub) (1:1000; Sigma-Aldrich), chicken anti-Glial Fibrillary Acidic Protein (GFAP) (1:4000; Abcam), mouse anti-Synaptophysin (1:500; Sigma-Aldrich), chicken MAP2 (1:4000; Novus), and chicken β-tub (1:4000; Abcam) were used together with secondary antibodies Alexa 488 donkey anti-rabbit, Alexa 568 donkey anti-mouse and Alexa 647 goat anti-chicken (all 1:400; Invitrogen). In addition, the nuclei of the cells were stained with 4',6-diamidino-2 phenylindole (DAPI), which was included in the mounting medium (Prolong Gold, Molecular Probes). The cells were imaged with a fluorescence microscope (Olympus IX51, Olympus Corporation) and a laser scanning confocal microscope (LSM 780, Carl Zeiss).

Ryynänen T., Toivanen M., Salminen T., Ylä-Outinen L., Narkilahti S, & Lekkala J. (2018). Ion Beam Assisted E-Beam Deposited TiN Microelectrodes—Applied to Neuronal Cell Culture Medium Evaluation. Frontiers in Neuroscience, 12, 882.

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Comprehensive Immunohistochemistry Panel for Neurodegenerative Research

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Rabbit anti-p53 (Santa Cruz Biotechnology), rabbit anti-p-JNK (Invitrogen), mouse anti-synaptophysin (Sigma), mouse anti-amyloid clone DE2B4 (Abcam), rabbit anti-amyloid clone FCA3542 (Calbiochem), mouse anti-amyloid MOAB2 (Novus; NBP2-13075)), pan anti-Tau k9JA (DAKO); mouse anti-p-Tau clone AT-8 (Thermo scientific), mouse anti-p-Tau clone PHF1 (a gift from Dr. Davies, Albert Einstein College of Medicine), mouse anti-NeuN (Chemicon), rabbit anti-cleaved caspase-3 (Cell Signaling), rabbit anti-GFAP (Dako; Z0334), Iba1 (Wako; 019-19741), mouse anti-Bmi1 clone F6 (Millipore), mouse anti-Bmi1 (Abcam), mouse anti-p-ATM (Novus), rabbit anti-p-ATR (Santa Cruz Biotechnology), rabbit anti-H3K9^me3 (Abcam), mouse anti-H2Aub clone E6C5 (Millipore), mouse anti-HP1 (Millipore), mouse anti-β-actin (Sigma), mouse anti-tubulin (Sigma), mouse anti-H3 (Upstate), and rabbit anti-mouse IgG (Upstate).

El Hajjar J., Chatoo W., Hanna R., Nkanza P., Tétreault N., Tse Y.C., Wong T.P., Abdouh M, & Bernier G. (2019). Heterochromatic genome instability and neurodegeneration sharing similarities with Alzheimer’s disease in old Bmi1+/− mice. Scientific Reports, 9, 594.

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Immunostaining and Thioflavin-S Staining Protocol

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Epitopes were demasked in either citrate (10 mM Tris-Na Citrate, pH 6.0) or EDTA (10 mM Tris Base, 1 mM EDTA, 0.05% Tween-20, pH 9.0) antigen retrieval buffer, and immunostained using the Dako Autostainer Plus automated slide processor and EnVision Flex system (Dako, Burlington, ON, Canada). Following deparaffinization and rehydration, sections were peroxidase treated, blocked in serum-free protein block, and immunostained with the following antibodies diluted in EnVision Flex Antibody Diluent: 1:2000 rabbit anti-Iba1 (Wako 019-19741, Richmond, VA, USA), 1:8000 rabbit anti-GFAP (Dako Z-0334), 1:2000 rabbit anti-Aβ_1-40 (F25276, laboratory developed), and 1:1000 mouse antisynaptophysin (Sigma-Aldrich S5768). Sections were treated with the kit-supplied appropriate mouse or rabbit HRP-conjugated secondary antibody and visualized with DAB. Sections were hematoxylin counterstained, dehydrated, and coverslipped with Permount (Fisher Scientific). Sections were digitally scanned with MIRAX SCAN for analysis (Zeiss, Don Mills, ON, Canada). After deparaffinization and rehydration, slides were stained with filtered 1% aqueous Thioflavin-S (Sigma-Aldrich).

Flores J., Noël A., Foveau B., Lynham J., Lecrux C, & LeBlanc A.C. (2018). Caspase-1 inhibition alleviates cognitive impairment and neuropathology in an Alzheimer’s disease mouse model. Nature Communications, 9, 3916.

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Endocytosis Assay for Synaptic Vesicle Recycling

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The endocytosis assay to monitor SV recycling was performed using rabbit polyclonal antibodies directed against the intravesicular domain of synaptotagmin1 (Synaptic System), applied for 5 min at RT on the cultures, as described previously [30 (link)]. Incubations with the antibody (1:400) were performed in Tyrode solution containing 124 mM NaCl, 5 mM KCl, 2 mM MgCl₂, 30 mM glucose, 25 mM HEPES, pH 7.4 and 2 mM CaCl₂. After fixation and permeabilization, a synaptophysin counter staining with mouse anti synaptophysin, 1:400 (Sigma-Aldrich) visualized the totality of synaptic vesicles. Acquired images were processed and quantitatively analyzed with ImageJ software as previously described [47 (link)]. Briefly, cultures were infected at DIV4 with GPF expressing viruses and assayed at DIV14 as in [22 (link)]. GFP positive processes were manually tracked and the number of synaptotagmin and synaptophysin positive clusters and synaptophysin positive clusters present in the region of interest were automatically counted.

Belluzzi E., Gonnelli A., Cirnaru M.D., Marte A., Plotegher N., Russo I., Civiero L., Cogo S., Carrion M.P., Franchin C., Arrigoni G., Beltramini M., Bubacco L., Onofri F., Piccoli G, & Greggio E. (2016). LRRK2 phosphorylates pre-synaptic N-ethylmaleimide sensitive fusion (NSF) protein enhancing its ATPase activity and SNARE complex disassembling rate. Molecular Neurodegeneration, 11, 1.

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Immunocytochemistry of 3D Cell Cultures

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3D cultures were fixed with 4% paraformaldehyde (PFA) for 1 h, washed 3 times with PBS and incubated for 2 h with blocking buffer (1% BSA, 5% goat serum, 0.15% saponin (Sigma Aldrich)). Samples were incubated 48 h with primary antibodies (1:200 mouse anti-MAP2 (Sigma Aldrich); 1:200 rabbit anti-GFAP (Dako) and 1:200 mouse anti-Olig1 (Millipore), 1:200 mouse anti-Synaptophysin (Sigma), 1:200 rabbit anti-PSD95 (Life Technologies), 1:1500 β-III-tubulin (Sigma Aldrich), 1:200 anti O4 (R&D Systems) diluted in blocking buffer) at 4 °C, followed by three washing steps and incubation with secondary antibodies (1:500 goat anti-mouse Alexa fluor 488 or 1:500 goat anti-rabbit Alexa fluor 568, diluted in blocking buffer, Molecular Probes) overnight. Then, samples were washed and incubated with Hoechst 33342 (1:10,000, Molecular Probes) for at least 1 h at room temperature (RT). After three washing steps, the samples were mounted on glass slides with Prolong Gold-antifade reagent (Molecular Probes) for confocal microscopy. Z-stacks started at the top of the sample were taking. Images were obtained with Zeiss LSM-510 (Zeiss) and Leica SP5 (Leica) confocal microscopes with identical time exposure and image settings.

Leite P.E., Pereira M.R., Harris G., Pamies D., dos Santos L.M., Granjeiro J.M., Hogberg H.T., Hartung T, & Smirnova L. (2019). Suitability of 3D human brain spheroid models to distinguish toxic effects of gold and poly-lactic acid nanoparticles to assess biocompatibility for brain drug delivery. Particle and Fibre Toxicology, 16, 22.

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Synaptic Inputs on KNDy Neurons

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In order to examine synaptic inputs onto KNDy neurons, NKB, was used as a cell marker since KNDy cells co-express all three peptides (kisspeptin, NKB and dynorphin) [28 (link)]. Immunolabeling and analysis was performed on 4 middle arcuate sections/animal/treatment. Sections were incubated with mouse anti-synaptophysin (1: 200, 17 h, Sigma Aldrich catalog item: S57689) followed by incubation in DyLight 650 goat anti-mouse (1:100, 30 min, Thermo Fisher catalog item 84545). Sections were next incubated overnight in rabbit anti-vGlut2 (1:15,000, 17 h, Synaptic Systems catalog item 135–402). Sections were then incubated sequentially with: 1) biotinylated goat anti-rabbit IgG (1:500, 1 h, catalog item BA-9200; Vector Laboratories), 2) Avidin-Biotin Complex reagent (1:500, 1 h, ABC, catalog item: pk-6100; Vector Laboratories), 3) biotinylated tyramine (1:250, 10 min, Tissue Sample Amplification (TSA), catalog item: NEL700A; PerkinElmer Life Sciences) and 4) Cy3-conjugated streptavidin (1:200, 30 min; Jackson ImmunoResearch, catalog item: 016–160-084). Finally, sections were incubated with guinea pig anti-proneurokinin B (1:1,000, 17h, gifted by Ciofi, IS-3/63, Bleed 210493) followed by DyLight 488 goat anti-guinea pig (1:100, 30 min, Thermo Scientific, catalog item SA5–10094).

Porter D.T., Goodman R.L., Hileman S.M, & Lehman M.N. (2021). Evidence that synaptic plasticity of glutamatergic inputs onto KNDy neurons during the ovine follicular phase is dependent on increasing levels of estradiol. Journal of neuroendocrinology, 33(3), e12945.

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Immunohistochemical Analysis of Neuronal Markers

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Dulbecco’s modified Eagle’s medium (DMEM), supplemented with fetal bovine serum (FBS) and penicillin–streptomycin, was provided by Invitrogen (Carlsbad, CA, USA). Goat polyclonal anti-doublecortin (DCX) and rabbit polyclonal anti-phospho-cAMP-response-element-binding protein (CREB) (pCREB) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Biotinylated horse anti-goat antibody, biotinylated goat anti-rabbit antibody, and avidin–biotin complex (ABC) were purchased from Vector Labs (Burlingame, CA, USA). Paraformaldehyde (PFA), 3,3-diaminobenzidine (DAB), sucrose, phosphate-buffered saline (PBS), 30% hydrogen peroxide (H₂O₂), lipopolysaccharide (LPS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), piracetam, dibutylphthalate polystyrene xylene (DPX) histomount medium, and mouse anti-synaptophysin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cerebrolysin was purchased from Ever Neuro Pharma (GMBH, Austria). The enzyme-linked immunosorbent assay (ELISA) development kit for NGF was purchased from R&D Systems (Minneapolis, MN, USA).

Choi J.G., Khan Z., Hong S.M., Kim Y.C., Oh M.S, & Kim S.Y. (2020). The Mixture of Gotu Kola, Cnidium Fruit, and Goji Berry Enhances Memory Functions by Inducing Nerve-Growth-Factor-Mediated Actions Both In Vitro and In Vivo. Nutrients, 12(5), 1372.

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