Ab4680

Manufactured by Abcam

Sourced in United Kingdom

Ab4680 is a reagent used in laboratory settings. It functions as a tool for research and experimentation purposes. The core purpose of this product is to provide a specific chemical component that can be utilized in various scientific applications.

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Lab products found in correlation

Ab40390, by Abcam (4 mentions) Ab6992, by Abcam (3 mentions) Ab75810, by Abcam (3 mentions) Ab7260, by Abcam (3 mentions) Ab15580, by Abcam (3 mentions) Alexa fluor 488, by Thermo Fisher Scientific (3 mentions) Ab150135, by Thermo Fisher Scientific (2 mentions) Ab5076, by Abcam (2 mentions) Mab377x alexa488, by Merck Group (2 mentions) Ap194c, by Thermo Fisher Scientific (2 mentions) Axio vision observer microscope, by Zeiss (2 mentions) Permafluor, by Thermo Fisher Scientific (2 mentions) Ab78078, by Abcam (2 mentions) Ab150073, by Abcam (2 mentions) Ab228549, by Abcam (2 mentions) Ab150088, by Abcam (2 mentions) Ab31851, by Abcam (2 mentions) Ab197485, by Abcam (2 mentions) Hoechst 33342, by Thermo Fisher Scientific (2 mentions) Eclipse 80i, by Nikon (2 mentions)

22 protocols using ab4680

Neurotoxin and Antibody Protocol

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α-LTx (LSP-130) and µ-conotoxin GIIIB (C-270) were purchased from Alomone. α-LTx purity was checked by SDS–PAGE, and its neurotoxicity by ex vivo mouse nerve-hemidiaphragm preparations, as previously described [28 (link)]. BoNT/A (Xeomin) was from Merz. All other reagents were from Sigma unless stated otherwise.
Antibodies and fluorescent conjugates with relative dilutions: α-BTx AlexaFluor555 (B35451 Thermo Fisher, 1:200), anti-Ctgf neutralizing antibody (70R-CR023 Fitzgerald, 2 µg/40 µl), anti-Ctgf for immunostaining (ab6992 Abcam, 1:200), anti-S100 (Z0311 Dako, 1:400), anti-GAP43 (ab75810 Abcam, 1:200), anti-NF (ab4680 Abcam, 1:800), anti-VAMP1 [29 (link)], anti-SNAP25 (ab24737 Abcam, 1:200), anti-SNAP-25 BoNT/A-cleaved [30 (link)], anti-syntaxin 1A/1B [31 (link)], anti-YAP (13008S, Cell Signaling, 1:200). Secondary AlexaFluor-conjugated antibodies (1:200) were from Thermo Fisher. A list of antibodies and the relative description is provided as Additional file 9.

Negro S., Lauria F., Stazi M., Tebaldi T., D’Este G., Pirazzini M., Megighian A., Lessi F., Mazzanti C.M., Sales G., Romualdi C., Fillo S., Lista F., Sleigh J.N., Tosolini A.P., Schiavo G., Viero G, & Rigoni M. (2022). Hydrogen peroxide induced by nerve injury promotes axon regeneration via connective tissue growth factor. Acta Neuropathologica Communications, 10, 189.

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Comprehensive Immunolabeling Protocol

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Primary antibodies against VIP (Abcam, ab78536), PACAP (Abcam, ab216627), VPAC1 (Santa Cruz, sc-30019; Abcam, ab123517), VPAC2 (Santa Cruz, sc-30020), PAC1 (Santa Cruz, sc-30018), myelin protein zero (Mpz) (Sigma, SAB2500665), myelin basic protein (Mbp) (Abcam, ab40390), neurofilament heavy chain (NF) (Abcam, ab4680), F4/80 (Abcam, ab6640), and GAPDH (EMD Millipore, MAB374) were used. Hoechst and species specific secondary antibodies conjugated with Alexa Fluor 488 or 568 dyes were purchased from Invitrogen. Horseradish peroxidase conjugated secondary antibodies for western blotting were purchased from Sigma.

Woodley P.K., Min Q., Li Y., Mulvey N.F., Parkinson D.B, & Dun X.P. (2019). Distinct VIP and PACAP Functions in the Distal Nerve Stump During Peripheral Nerve Regeneration. Frontiers in Neuroscience, 13, 1326.

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Multimodal Visualization of Rat Brain

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A fixed brain sample from a 6 month-old rat was cut into 30 μm-thick slices using a cryomicrotome, and positioned on glass slides. Then, immunohistochemical stainings were performed to label various microstructure features. After a step of blocking non-specific antigens with donkey serum 5% buffer, with detergent (Triton, Sigma Aldrich, X-100, 1% 2 h incubation), a quadruple staining was prepared. It included labeling for microglial cells (anti-Iba 1, AbCam ab5076, 1/500 dilution), astrocytes (anti-GFAP, AbCam, ab7260, 1/500 dilution), neuron micro-filaments (anti-NF, AbCam ab4680, 1/2000 dilution) and neuron nuclei (anti-NeuN, Millipore, MAB377X Alexa488, 1/100 dilution). Each antibody was incubated for one hour, followed by two steps of washing with PBS. Secondary antibodies were incubated at the same time, with anti-NeuN already coupled with Alexa488. We used donkey anti-chicken Cy5 (Millipore, AP194C), donkey anti-rabbit Alexa350 (ThermoFisher, 1710039) and donkey anti-goat Alexa647 (AbCam, ab150135).
Slices were mounted with Permafluor (ThermoFisher, TA-030-Fr), then fluorescence microscopy images acquired with an Axio Vision Observer microscope at x20 magnification (Carl Zeiss).
The patterns of staining intensity across the brain (mainly cortex and hippocampus) were compared to patterns of NEXI model parameters, in particular neurite density

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Jelescu I.O., de Skowronski A., Geffroy F., Palombo M, & Novikov D.S. (2022). Neurite Exchange Imaging (NEXI): A minimal model of diffusion in gray matter with inter-compartment water exchange. NeuroImage, 256, 119277.

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Immunofluorescence Markers of Neural Tissue

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The following primary antibodies were used for IF at the indicated dilutions: anti-P0 (1/500, Abcam, ab39375), anti-p75 (1/500, Millipore, ab1554), anti-S100 (1/1000, Dako, Z0311), anti-Iba1 (1/500, Wako, 019-19741), anti-CD31 (1/100, BD Biosciences, 553370), anti-neurofilament 200 kD (1/1000, Abcam, ab4680), anti-laminin (1/500, Abcam, 11575), anti-collagen III (1/1000, Abcam, ab7778), anti-fibronectin (1/500, Sigma-Aldrich, clone FN-3E2), anti-NG2 (1/500, Millipore, ab5320), anti-PDGFRβ (1/500, Abcam, ab32570), anti-αSMA (1/1000, Sigma-Aldrich, C6198), anti-GFP (1/1000, Abcam, ab13970), anti-Glut1 (1/500, Abcam, ab652), anti-F4/80 (1/100, Bio-Rad, MCA497G) and anti-NG2 (1/100, Thermo Fisher Scientific, MA5-24247). For further details of primary antibodies used in this study, see Table S1.

Stierli S., Napoli I., White I.J., Cattin A.L., Monteza Cabrejos A., Garcia Calavia N., Malong L., Ribeiro S., Nihouarn J., Williams R., Young K.M., Richardson W.D, & Lloyd A.C. (2018). The regulation of the homeostasis and regeneration of peripheral nerve is distinct from the CNS and independent of a stem cell population. Development (Cambridge, England), 145(24), dev170316.

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Immunofluorescence Labeling of Neurons

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Primary antibodies including neurofilament 200 (NF200, ab4680), glial fibrillary acidic protein (GFAP, ab7260), NeuN (ab104224), growth associated protein 43 (GAP43, ab75810), and LC3-II (ab192890), and secondary antibodies including goat anti-mouse 488 (ab150113), goat anti-rabbit 488 (ab150077), goat anti-chicken 488 (ab150169) and goat anti-rabbit tritc (ab6718) were purchased from Abcam (MC, United Kingdom). The DAPI was also under the supply of Abcam (MC, United Kingdom). Recombinant human FGF21 (rhFGF21) was obtained from Prof. Xiaokun Li, Zhejiang Provincial Key Laboratory of Biopharmaceuticals, Wenzhou Medical College, Wenzhou, Zhejiang, China. It was previously reported that rhFGF21 was produced using Escherichia coli and purified to be endotoxin free (Wang et al., 2010 (link)), and its biological effect further tested in nerve cells (Lu et al., 2019 (link)).

Zhu S., Ying Y., Ye L., Ying W., Ye J., Wu Q., Chen M., Zhu H., Li X., Dou H., Xu H., Wang Z, & Xu J. (2021). Systemic Administration of Fibroblast Growth Factor 21 Improves the Recovery of Spinal Cord Injury (SCI) in Rats and Attenuates SCI-Induced Autophagy. Frontiers in Pharmacology, 11, 628369.

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Immunostaining of Murine DRG Neurons

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Male adult mice were euthanized by CO₂ and decapitated, DRGs were dissected out and postfixed in 4% PFA for 1 h at 4 °C. Samples were subsequently washed in PBS and cryopreserved at 4 °C overnight with 30% sucrose in PBS. Tissue was then embedded in OCT and frozen at −20 °C. Samples with 10 μm sections were cut on a freezing sledge microtome (Leica Microsytems) and air dry in room temperature for two hours to prepare for staining. Two mice were used for each immunostaining, showing similar results. Sections were permeabilized in PBS+0.5%Triton-x100 for 10 min. Following blocking in 1%BSA PBS for 1 hour, samples were then incubated in primary antibody, including chicken anti-NEFH (abcam, ab4680), rabbit anti-PTEN (abcam, ab32199), rabbit anti-c-JUN(abcam, ab31419), rabbit anti-STAT5B (abcam, ab76319), rabbit anti-PDCD2 (Proteintech,10725-1-AP) at room temperature for 2 hours. For detection, Donkey 488- and Cy3-conjugated Alexa secondary against rabbit and goat were used at dilution of 1:400. (Jackson ImmunoResearch 705-545-003 and 703-166-155) for one hour in the room temperature. After PBS washes, slides were subsequently mounted with mounting medium (darko) and visualized with a Nikon Fluorescent microscope.

Hu G., Huang K., Hu Y., Du G., Xue Z., Zhu X, & Fan G. (2016). Single-cell RNA-seq reveals distinct injury responses in different types of DRG sensory neurons. Scientific Reports, 6, 31851.

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Immunofluorescence and Western Blot Antibody Protocol

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The following primary antibodies were used for immunofluorescence staining at the indicated dilutions: neurofilament (NF200) heavy 200 kD (1:500; ab4680; Abcam), cJun (1:200; 9165S; Cell Signaling Technology), Iba1 (1:300; 019–19741; Wako Pure Chemical Industries), F4/80 (1:300; ab16911; Abcam), and Ki67 (1:200; ab15580; Abcam).
The following primary antibodies were used for Western blotting: myelin protein zero (1:1,000; SAB2500665; Sigma-Aldrich), MBP (1:2,000; sc-13912; Santa Cruz Biotechnology, Inc.), Krox20 (1:500; PRB-236P-100; Covance), β-tubulin (1:2,000; sc-134229; Santa Cruz Biotechnology, Inc.), cJun (1:1,000; 9165; Cell Signaling Technology), p-cJun (1:500; 9261; Cell Signaling Technology), p-Erk1/2 (1:2,000; 9101; Cell Signaling Technology), p-p38 (1:500; 4631; Cell Signaling Technology), YAP/TAZ (1:1,000; 8418; Cell Signaling Technology), TAZ (1:1,000; 4883; Cell Signaling Technology), YAP (1:1,000; 14074; Cell Signaling Technology), connective tissue growth factor (1:1,000; ab6992; Abcam), collagen IV (1:1,000; ab6586; Abcam), MCP1 (1:500; 500-P113; PeproTech), active β-catenin (1:1,000; 05–665; EMD Millipore), N-cadherin (1:1,000; ab76057; Abcam), E-cadherin (1:1,000; PharMingen Clone 36 610181; BD), p-Akt (1:2,000; 4060; Cell Signaling Technology), and vinculin (1:1,000; 4650; Cell Signaling Technology).

Mindos T., Dun X.P., North K., Doddrell R.D., Schulz A., Edwards P., Russell J., Gray B., Roberts S.L., Shivane A., Mortimer G., Pirie M., Zhang N., Pan D., Morrison H, & Parkinson D.B. (2017). Merlin controls the repair capacity of Schwann cells after injury by regulating Hippo/YAP activity. The Journal of Cell Biology, 216(2), 495-510.

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Immunostaining of SH-SY5Y Cells

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SH-SY5Y cells were fixed in 4% paraformaldehyde for 20 min, and permeabilized with PBS containing 0.05% Triton X-100 and 5% goat serum for 60 min at room temperature. Permeabilization samples were incubated with primary antibodies of neurofilament-H (NF-H) (ab4680, abcam), beta III Tubulin (TUBB3) (ab78078, abcam), NDRG3 (ab133715, abcam) at 4 °C overnight. Immunostaining and nuclei were visualized by Alexa Fluor-488 (A-11001, ThermoFisher Scientific) or Alexa Fluor-555 (A-21428, ThermoFisher Scientific) fluorescence-conjugated secondary antibodies and Hoechst-33342 (H3570, ThermoFisher Scientific).

Xu Y., Kusuyama J., Osana S., Matsuhashi S., Li L., Takada H., Inada H, & Nagatomi R. (2023). Lactate promotes neuronal differentiation of SH-SY5Y cells by lactate-responsive gene sets through NDRG3-dependent and -independent manners. The Journal of Biological Chemistry, 299(6), 104802.

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Immunostaining and Western Blotting Assay

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Primary antibodies used for immunostaining were: Neurofilament (1:1000; ab4680; Abcam), ALDH1A1 (1:200; ab52492; Abcam), YAP (1:100; #14074; CST), TAZ (1:100; sc-48805; Santa Cruz), Ki67 (1:100; ab15580; Abcam), S100 (pre-diluted; GA504; Dako). Species-specific AlexaFluor™ secondary antibodies (Thermo Fisher) were used at 1:200.
Primary antibodies used for western blotting: YAP (1:1000; #14074; CST), TAZ (1:500; sc-48805; Santa Cruz), Pan-TEAD (1:1000; #13295; CST), GAPDH (1:5000; AB2302; Merck), ALDH1A1 (1:1000, ab52492; Abcam) and CTGF (1:500, ab6992, Abcam). For detection of primary antibodies, HRP-conjugated goat anti-rabbit (1:5000; #1706515; Bio-Rad) and HRP-goat anti-mouse (1:5000; #1721011; Bio-Rad) were used.

Laraba L., Hillson L., de Guibert J.G., Hewitt A., Jaques M.R., Tang T.T., Post L., Ercolano E., Rai G., Yang S.M., Jagger D.J., Woznica W., Edwards P., Shivane A.G., Hanemann C.O, & Parkinson D.B. (2022). Inhibition of YAP/TAZ-driven TEAD activity prevents growth of NF2-null schwannoma and meningioma. Brain, 146(4), 1697-1713.

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Immunofluorescent Labeling of Tissue and Cells

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The immunofluorescent process of tissue or cells was done as previously described.40, 43 The detailed information of primary and secondary antibodies was shown as following: NF‐200 (1:10000, Abcam, ab4680), MBP (1:200, Abcam, ab40390), HO‐1 (1:100, Santa Cruz, sc‐1797), NQO1 (1:1000, Abcam, ab34173), S100 (1:200, Abcam, ab4066), Nrf‐2 (1:500, Abcam, ab62352), Alexa‐Fluor 488 donkey anti‐ rabbit IgG (1:1000, Abcam, ab150073), and Alexa‐Fluor 594 donkey anti‐mouse IgG (1:1000, Abcam, ab150108). Nuclei were labelled with 4′6‐Diamidino‐2‐phenylindole‐dihydrochloride (DAPI, C1006; Beyotime Institute of Biotechnology, Shanghai, China). All fluorescence images were obtained under the Nikon ECLIPSE 80i (Nikon, Tokyo, Japan).

Lu Y., Li R., Zhu J., Wu Y., Li D., Dong L., Li Y., Wen X., Yu F., Zhang H., Ni X., Du S., Li X., Xiao J, & Wang J. (2018). Fibroblast growth factor 21 facilitates peripheral nerve regeneration through suppressing oxidative damage and autophagic cell death. Journal of Cellular and Molecular Medicine, 23(1), 497-511.

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