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Anti synaptophysin antibody

Manufactured by Abcam
Sourced in United Kingdom, United States

Anti-synaptophysin antibody is a laboratory reagent used for the detection and localization of synaptophysin, a protein found in synaptic vesicles. It is commonly used in immunohistochemistry and Western blotting techniques to study the distribution and expression of synaptophysin in various tissues and cell types.

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10 protocols using anti synaptophysin antibody

1

Neuronal Differentiation and Neuroprotection

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DMEM/F12 and B27™ culture media were bought from GIBCO® (U.S.A.) and trypsin (no. 0458) was bought from Amresco (U.S.A.). Poly-l-lysine (PLL), laminin and nerve growth factor (NGF) were purchased form Sigma (St. Louis, MO). Harpagide was obtained from Hubei Jusheng Technology Co., Ltd (Wuhan, China). Anti-α-synuclein antibody, anti-α-synuclein (phospho S129) antibody and anti-synaptophysin antibody were purchased from Abcam (U.K.). Anti-tyrosine hydroxylase (TH) was purchased from and the fluorescent secondary antibodies were purchased from Wuhan Boster Corp (China). Reactive oxygen species (ROS) assay kit and cell counting kit-8 (CCK-8) assay were purchased from Beyotime Biotechnology Ltd. (China) and Dojindo Laboratories (Japan), respectively. All other chemicals unless specified were reagent grade and were used without further purification.
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2

Histological Analysis of Implanted Constructs

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After formalin-fixation, the constructs were cut perpendicular to the axis of the AV loop. The halves of the constructs were paraffin embedded. The half including the vessels entering the loop was referred to as “proximal” while the other half, containing the vein graft was referred to as “distal”. 3 µm cross-sections were cut of each half and stained with hematoxylin and eosin (HE) and Masson’s Trichome according to standard protocols. Vessels were visualized with α-smooth muscle actin (SMA) staining (primary antibody: mouse monoclonal antibody actin smooth muscle, Zytomed Systems, Berlin, Germany; secondary antibody: conjugation of anti-mouse (Ig) and anti-Rabbit (Ig), Life Technologies, Carlsbad, CA, USA) [22 (link)]. Methylenblau, Synaptophysin immunofluorescence (anti-Synaptophysin antibody, Abcam, Cambridge, UK) and S100 immunohistochemial (S100 monoclonal antibody, Life Technologies) stainings were used for visualization of the implanted nerve. For macrophage detection, including pro- (M1-) and anti-inflammatory (M2-) macrophages CD86 (Abcam) and CD163 (Leica Biosystems Inc., Deer Park, IL, USA), immunostainings were performed as previously described [21 (link)]. After completing the staining, all sections were photographed under 10× magnification, using an Olympus IX81 (Olympus, Hamburg, Germany), and then stitched together, using the software cellSens Dimension V.1.5.
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3

Quantification of Neuromuscular Junctions in EDL Muscle

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The extensor digitorum longus (EDL) muscle was split into four pieces and fixed with 4% PFA for 30 min. After washing with phosphate-buffered saline (PBS), the muscles were blocked by incubating overnight in a blocking solution consisting of 1% Triton X-100 and 4% BSA in PBS at 4 °C, followed by incubation for 1 day with anti-synaptophysin antibody (1:200; Abcam, #ab14692) diluted with the blocking solution at 4 °C with rotation. Subsequently, the muscles were incubated for 1 day with Alexa Fluor 488 α-Bungarotoxin (1:1000; Thermo Fisher, #1313422) and Alexa Fluor 594 anti-rabbit IgG (1:1000; Jackson ImmunoResearch, #711-585-152) diluted with the blocking solution at 4 °C with rotation. The stained muscles were counterstained with DAPI (Dojindo) and mounted with SlowFade Gold antifade reagent (Thermo Fisher). Z-stack images were captured using the confocal laser scanning microscope system TCS SP8 (Leica).
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4

Protein Expression Analysis in Brain Tissues

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Western blot analysis was performed as described [21 (link)]. Tissues were harvested from control, S-W, and MSC groups (12 hours before surgical wound) at 6 hours after surgical wound. Anti-Claudin-5 antibody (Invitrogen, USA), anti-Occludin antibody (Abcam, USA), anti-ZO-1 antibody (Abcam, USA), anti-matrix metalloproteinase-9 (MMP-9) antibody (Abcam, USA), anti-synaptophysin antibody (Abcam, USA), and anti-endothelial nitric oxide (eNOS) antibody (Abcam, USA) were performed in the analysis. The ratio of the levels of targeted proteins to the level of β-actin was performed to analyze the trend of those proteins in the brain tissues.
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5

Hippocampus Protein Expression Analysis

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Western blot analysis was performed using the methods described in our previous studies [63 (link)]. Hippocampus tissues were harvested from the mice at 11 days after the anesthesia/surgery or control condition. We used postsynaptic density protein (PSD)-95 antibody (1:1,000, molecular weight of 95 kDa, Cell Signaling, Danvers, MA) to detect PSD-95 level. We used anti-synaptophysin antibody (1:1,000, molecular weight of 37, Abcam, Cambridge, MA) to detect the synaptophysin levels. An β-Actin antibody (1:5,000, molecular weight of 42 kDa, Sigma, St. Louis, MO) was used to detect non-targeted protein β-Actin. β-Actin amount was used to normalize (e.g., determining the ratio of PSD-95 to β-Actin amount) protein levels and control for loading differences in the total protein amount. The quantification of the Western blot was performed as described in previous study [63 (link)]. We presented changes in protein levels in the hippocampus of the mice received the anesthesia/surgery as a percentage of those in the mice in the control group. 100% of protein level changes referred to control levels for comparison to experimental conditions. Signal intensity was analyzed using a Bio-Rad (Hercules, CA) image program.
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6

Western Blot Analysis of Neuronal and Glial Markers

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Protein extracts were separated by 10 % (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred to polyvinylidene difluoride membrane. The membrane was incubated with following primary antibodies for anti-total p38/JNK/ERK MAPK, anti-phospho-p38/JNK/ERK MAPK antibodies (1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-Synaptophysin antibody (1:1000, Abcam), anti-PSD95 antibody (1:1000, Abcam), anti-NeuN antibody (1:1000, Millipore), anti-MAP2 antibody (1:1000, Abcam), anti-GFAP antibody (1:5000, Dako, Glostrup, Denmark), anti-KCa3.1 antibody (1:400, Abcam), and anti-β-actin antibody (1:3000, Sigma). Secondary antibodies were horseradish peroxidase-conjugated antibody (1:3000; Amersham Biosciences) for 1 h at room temperature.
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7

Immunofluorescence Analysis of Brain Tissues

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For IF analysis of tissues, paraffin-embedded brain sections were treated by deparaffinization and antigen retrieval. After blocking with the serum of goat or horse or bovine serum albumin, the samples were incubated with primary antibodies overnight at 4°C. Sections were stained with anti-Qki antibody (rabbit, 1:200; Bethyl Laboratories); anti-Iba1 antibody (goat, 1:500; Abcam); anti-GFP antibody (chicken, 1:200; Abcam); anti-Mbp antibody (mouse, 1:200; Covance); anti-Olig2 antibody (mouse, 1:200; EMD Millipore); anti-PDGFRα-2 antibody (rabbit, 1:200; Cell Signaling Technology); anti-GSTπ antibody (1:200; MBL International); anti-synaptophysin antibody (rabbit, 1:200; Abcam); and anti-APP antibody (1:500; Covance). The samples were then incubated with species-appropriate secondary antibodies from goat or donkey coupled with AlexaFluor dyes (488, 568, or 594; Thermo Fisher Scientific) for 1 h at room temperature at a dilution of 1:1,000. VECTASHIELD with DAPI (Vector Laboratories) was used to mount coverslips. IF images were taken with a Leica DMI8 inverted fluorescent microscope. For costaining, Z-stack images were taken. The colocolization analysis in Fig. 4 E was performed with the Leica LASX software package.
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8

Assessing Neuromuscular Junction Integrity

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Tibialis anterior muscle samples were dissected into thin bundles under a binocular microscope. Bundles were collected from at least three different parts of the muscle. Acetylcholine receptors in the postsynaptic apparatus of neuromuscular junctions were labeled with rhodamine-conjugated α-bungarotoxin (Sigma–Aldrich). Immunofluorescent labeling of nerve terminals was performed with a rabbit polyclonal anti-synaptophysin antibody diluted 1/200 (Abcam, Cambridge, UK), and Alexa-conjugated goat anti-rabbit IgG diluted 1/500 (Jackson ImmunoResearch, Suffolk, UK). Muscle bundles were mounted onto slides, prior to fluorescence microscopy. Neuromuscular junctions were considered as denervated when the presynaptic nerve terminal was absent from the postsynaptic region. Integrity of the typical pretzel-shaped morphology of neuromuscular junctions was determined as previously described (63 (link)) by quantifying the number of separate postsynaptic gutters, which estimates the degree of fragmentation, and the number of gutter intersections, which estimates the degree of enlargement and complexity of the postsynaptic apparatus.
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9

Immunocytochemistry of Cultured Neurons

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Immunocytochemistry of CGC cultures was performed as described previously by Puigdellívol et al. (2015 (link)). Briefly, after 14 DIV, cells were washed with PBS and fixed with PBS containing 4% PFA for 10 min at room temperature. Cells were washed three times with PBS and blocked using PBS containing 0.1 M glycine for 10 min. After three washes with PBS, cells were permeabilized using PBS containing 0.1% saponin for 10 min, rinsed three times with PBS, and blocked using PBS containing 15% BSA for 30 min. Cells were then washed with PBS and incubated overnight at 4°C with PBS containing 5% BSA, and anti‐synaptophysin antibody (1:300, Abcam, 32127). The next day, cells were washed three times with PBS and incubated with PBS containing 5% BSA, and Alexa‐Fluor 488 anti‐rabbit antibody (1:200, Thermo Fisher, A11008) for 2 h at room temperature. Cells were then washed three times with PBS and mounted on glass slides using Vectashield mounting medium with DAPI (Vector Laboratories, H1500). Slides were stored at 4°C until analysis via confocal microscopy.
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10

Characterization of MWCNT Nanoparticles

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The MWCNTs used in this study were obtained from the Institute of Metal Research, China Academy. The average length of MWCNTs was approximately 2μm and the diameter was approximately 10-20nm. The MWCNTs were suspended in 0.9% NaCl with 0.1% Tween 80, and the suspensions were sonicated for 20 minutes before each use. The characteristics of the nanoparticles used in this study can be found in our previous study (Han et al., 2012) .
Anti-NMDAR2B antibody and anti-Synaptophysin antibody were purchased from Abcam (Cambridge, UK). Anti-LC3 antibody was obtained from MBL (Nagoya, Japan). Anti-Beclin-1 antibody was purchased from Cell Signaling Technology (MA, USA). Anti-β-actin antibody was purchased from Santa Cruz Biotechnology, Inc. CA (California, USA).
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