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Mouse monoclonal anti nestin

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Mouse monoclonal anti-Nestin is a laboratory reagent used for the detection and identification of the Nestin protein. Nestin is an intermediate filament protein that is expressed in neural stem and progenitor cells. This product can be used in various research applications that involve the study of Nestin and its expression patterns.

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Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions) Paraformaldehyde (pfa), by Merck Group (2 mentions) Alexa fluor 488, by Thermo Fisher Scientific (2 mentions) Lsm 780, by Zeiss (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Epidermal growth factor (egf), by Thermo Fisher Scientific (2 mentions) Goat anti mouse igg, by Jackson ImmunoResearch (1 mentions) Anti oct4, by Abcam (1 mentions) Alexaflour 488 or 594 secondary antibodies, by Thermo Fisher Scientific (1 mentions) Mouse monoclonal anti hif 1α, by Abcam (1 mentions) Mouse monoclonal anti mucin5ac, by Santa Cruz Biotechnology (1 mentions) Fv1000, by Olympus (1 mentions) Alexa fluor 488 anti mouse igg, by Thermo Fisher Scientific (1 mentions) Rabbit anti gfap, by Merck Group (1 mentions) Rabbit anti map2, by Merck Group (1 mentions) Rabbit anti ng2 chondroitin sulfate proteoglycan, by Merck Group (1 mentions) Mouse monoclonal anti βiii tubulin, by Promega (1 mentions) Triton x 100, by Merck Group (1 mentions) Goat serum, by Merck Group (1 mentions) Normal goat serum (ngs), by Jackson ImmunoResearch (1 mentions)

Close spelling variants of this product

Nestin (147 citations) Anti-Nestin (77 citations) Mouse anti-Nestin (56 citations) Mouse anti-nestin monoclonal antibody (11 citations) Monoclonal anti-Nestin (2 citations) Monoclonal mouse (2 citations) Mouse anti-rat anti-nestin monoclonal antibody (2 citations)

13 protocols using mouse monoclonal anti nestin

1

Immunofluorescence Staining of Neural Cells

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Fixed cells were washed 3 times for 5 minutes with PBS. Blocking was performed with Serum Blocking Solution (PBS contains 5% normal goat serum, 1% BSA, and 0.3% Triton X) at room temperature for 1 hour, and primary antibodies were added including mouse monoclonal anti-Nestin (1:200, Millipore), rabbit polyclonal anti-Glial Fibrillary Acidic Protein (GFAP, 1:400, Sigma), mouse monoclonal anti-beta-Tubulin Isotype III (1:800 Sigma)) and rabbit polyclonal anti-olig2 for oligodendroglial lineage cells (1:200, Millipore). Cells with primary antibodies were incubated at 4°C overnight.
The cells incubated with primary antibodies were washed 1 minute with PBS for 5 times, and secondary antibodies were added using goat Anti-Mouse IgG (Jackson ImmunoResearch Laboratories), or goat anti-rabbit (life technology Alexa fluor 488) at 1:200 with blocking solution at room temperature for 1 hour. Cells were washed 1 minute with PBS for 5 times. Nuclear counterstain was performed with DAPI (4',6-Diamidino-2-Phenylindole, Dilactate) at 1:1000 with blocking solution at room temperature for 15 min, washed 1 minute with PBS for 5 times, and mounted with Vectashield mounting media (Victor Laboratories). To avoid risk of cell loss, slides were sealed with nail polish (OPI RapiDry TopCoat).
Zhao H., Steiger A., Nohner M, & Ye H. (2015). Specific Intensity Direct Current (DC) Electric Field Improves Neural Stem Cell Migration and Enhances Differentiation towards βIII-Tubulin+ Neurons. PLoS ONE, 10(6), e0129625.
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2

Immunofluorescence Analysis of 3D Spheroids

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Spheroids were fixed with 4% paraformaldehyde for 24 hours, dehydrated through a graded series of sucrose, embedded in paraffin, and sectioned. Four-micron-thick sections were blocked with 10% goat serum for 30 minutes at room temperature then incubated with mouse monoclonal anti-HIF-1α (diluted 1:500; Abcam), mouse monoclonal anti-Nestin (diluted 1:200; Millipore, Bedford, MA), rabbit polyclonal anti-Oct4 (diluted 1:200; Abcam), mouse monoclonal anti-mucin5ac (diluted 1:100; Santa Cruz), or mouse monoclonal anti-mucin 6 (diluted 1:100; Santa Cruz, http://www.scbt.com/datasheet-71623-mucin-6-0-n-459-antibody.html) antibodies for 1 hour. After washing with PBS three times, sections were incubated with corresponding secondary Alexaflour 488 or 594 secondary antibodies (all diluted 1:200; Invitrogen, https://www.lifetechnologies.com/order/catalog/product/A14808?ICID=search-product). Nuclei were stained with DAPI (Sigma-Aldrich). Fluorescence staining was visualized using an immunofluorescence microscope (FV1000, Olympus, Japan). In all experiments, at least 10 spheroids per group were analyzed.
Miao Z.F., Wang Z.N., Zhao T.T., Xu Y.Y., Gao J., Miao F, & Xu H.M. (2014). Peritoneal Milky Spots Serve as a Hypoxic Niche and Favor Gastric Cancer Stem/Progenitor Cell Peritoneal Dissemination Through Hypoxia-Inducible Factor 1α. Stem Cells (Dayton, Ohio), 32(12), 3062-3074.
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3

Immunofluorescent Characterization of Neural Cell Types

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Immunostaining was performed as previously described (Wang et al. 2013 (link)). Briefly, cells were fixed in 4% (w/v) paraformaldehyde (Sigma) and permeabilized in 0.1% (v/v) Triton X-100 (Sigma). Non-specific antigenic sites were blocked with PBS-T containing 4% (v/v) goat serum (Sigma) and 1% (v/v) glycerol for 20 min at room temperature. Cells were immunostained with mouse anti-oligodendrocyte marker O4 (1:200, R&D system, Minneapolis, MN), rabbit anti-NG2 Chondroitin Sulfate Proteoglycan (1:200, Millipore, Billerica, MA), mouse monoclonal anti-nestin (1:1000, Millipore), mouse monoclonal anti-βIII-tubulin (1:1000, Promega, Madison, WI), rabbit anti-MAP2 (1:100 Sigma) and rabbit anti-GFAP (1:1000, Sigma) antibodies followed by corresponding secondary antibodies (anti-mouse IgG Alexa Fluor 488, 1:400; anti rabbit IgG Alexa Fluor 594, 1:400; anti-mouse IgM Alexa Fluor 488, 1:400; Invitrogen) and 4’,6-diamidino-2-phenylindole (DAPI, Sigma) nuclear staining. Images were acquired on an EVOS fluorescence microscope (AMG, Bothell, WA).
Choi E., Xu Y., Medynets M., Monaco M.C., Major E.O., Nath A, & Wang T. (2018). Activated T cells induce proliferation of oligodendrocyte progenitor cells via release of vascular endothelial cell growth factor-A. Glia, 66(11), 2503-2513.
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4

Immunofluorescence Staining of Neural Cells

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Cells were fixed for 10 minutes at room temperature with 4% paraformaldehyde, and then triple washed with phosphate-buffered saline for 5 minutes each time. Cells were permeabilized for 20 minutes at room temperature with 0.3% Triton X-100, and then blocked with 10% normal goat serum (Jackson Immunoresearch Laboratories, Burlington, ON, Canada) in phosphate-buffered saline for 1 hour at room temperature. Samples were processed with primary antibodies for NPCs (mouse monoclonal anti-Nestin, 1:400; Millipore, Etobicoke, ON, Canada), neurons (rabbit polyclonal anti-class III β-tubulin; Covance, Princeton, NJ, USA) and astrocytes (rabbit polyclonal anti-glial fibrillary acidic protein, 1:500; Sigma) overnight at 4°C. Samples were triple washed with phosphate-buffered saline for 5 minutes, and incubated with appropriate secondary antibodies for 1 hour at 37°C. Secondary antibodies used were Alexafluor 568 (goat anti-mouse, 1:400; Life Technologies), Alexafluor 488 (goat-anti-rabbit, 1:400; Life Technologies) and Alexafluor 488 (goat anti-mouse, 1:300; Life Technologies). Mounting medium containing 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlington, ON, Canada) was used for nuclear staining.
Babona-Pilipos R., Pritchard-Oh A., Popovic M.R, & Morshead C.M. (2015). Biphasic monopolar electrical stimulation induces rapid and directed galvanotaxis in adult subependymal neural precursors. Stem Cell Research & Therapy, 6(1), 67.
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5

Immunofluorescence Analysis of Human iPSCs

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The human iPSCs cultured in TCP or PS inserts were fixed by adding 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 15 minutes and then permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 5 minutes. After blocking with 3% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) solution for 30 minutes, the cells were incubated with mouse anti-human primary antibodies (mouse monoclonal anti-neuronal β-III Tubulin, mouse monoclonal anti-Nestin and mouse monoclonal anti-MAP2 (all are from Millipore, Darmstadt, Germany)) at 4 °C overnight. After washing with PBS, the secondary antibodies (Alexa Fluor-488 goat anti-mouse IgG, 1:500; Alexa Fluor-555 goat anti-mouse IgG, 1:500; Life Technologies, Darmstadt, Germany) were added and incubated for 60 minutes. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO, USA).
After 3 times of washing with PBS, the samples were visualized using a fluorescence microscope (AxioSkop, Carl Zeiss, Jena, Germany) or scanned with a confocal laser scanning microscope (LSM 780, Carl Zeiss, Jena, Germany).
For the cell characterization staining, the human iPSCs were stained with the Fluorescent Human ES/iPS Cell Characterization Kit (Millipore, Darmstadt, Germany) following the manufacturer's protocol.
Li Z., Wang W., Kratz K., Küchler J., Xu X., Zou J., Deng Z., Sun X., Gossen M., Ma N, & Lendlein A. (2016). Influence of surface roughness on neural differentiation of human induced pluripotent stem cells. Clinical hemorheology and microcirculation, 64(3).
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6

Immunohistochemical Analysis of Neurogenesis

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A series of coronal sections were selected to perform immunochemical staining of BrdU, nestin, DCX, NeuN, and laminin. The primary antibodies were applied including sheep polyclonal anti-BrdU (1 : 500, Abcam, USA), mouse monoclonal anti-BrdU (1 : 1000, Sigma-Aldrich, USA), mouse monoclonal anti-nestin (1 : 1000, Chemicon, USA), mouse monoclonal anti-neuronal nuclei (NeuN, 1 : 1000, Chemicon, USA), or rabbit polyclonal anti-laminin (1 : 200, Sigma-Aldrich, USA). After blocking with goat serum and incubation with primary antibodies overnight at 4°C, the slices were incubated with fluorescent-labeled secondary antibodies Alexa Fluor 488-conjugated anti-sheep IgG (1 : 200), Cy3-conjugated anti-mouse IgG (1 : 200), or Cy3-conjugated anti-rabbit IgG (1 : 200; all three antibodies from Jackson Immunoresearch Laboratories, USA) for 1 h at room temperature. For BrdU immunofluorescence, brain sections were pretreated for 30 min at 37°C with 2 N HCl and then rinsed for 10 min in 0.1 M boric acid (pH = 8.5) at room temperature followed by incubation with blocking solution. Phosphate-buffered saline instead of primary antibody was applied in negative control. The images were taken from four different fields at least (Olympus BX51; Olympus).
Chen L., Wang X., Zhang J., Dang C., Liu G., Liang Z., Huang G., Zhao W, & Zeng J. (2016). Tongxinluo Enhances Neurogenesis and Angiogenesis in Peri-Infarct Area and Subventricular Zone and Promotes Functional Recovery after Focal Cerebral Ischemic Infarction in Hypertensive Rats. Evidence-based Complementary and Alternative Medicine : eCAM, 2016, 8549590.
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7

Protein Expression Analysis in Vascular Cells

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Lysates (30-50 μg) were prepared from the carotid artery, aorta or VSMCs, subjected to SDS-polyacrylamide gel (10%) electrophoresis and transferred to a PVDF membrane (Perkin Elmer Life Sciences, Boston, MA) [16 ]. Antibodies used include a mouse monoclonal anti-nestin (1:500; Chemicon), mouse monoclonal anti-eNOS (1:500; BD Bioscience), goat monoclonal anti-CD31 (1:500; SantaCruz Biotechnologies), rabbit polyclonal anti-caldesmon (1:2500; Abcam), rabbit polyclonal anti-smooth muscle-22α (1:5000; Abcam), rabbit polyclonal anti-smooth muscle α-actin (1:5000; Abcam), and mouse monoclonal anti-GAPDH (1:50,000; Ambion, Austin TX). Following overnight incubation at 4°C, the appropriate secondary antibody-conjugated to horseradish peroxidase (1:20,000, Jackson Immunoresearch, West Grove, PA) was added and bands visualized utilizing the ECL detection kit (Perkin Elmer). Films were scanned with Image J software® and the target protein signal was depicted as arbitrary light units normalized to GAPDH protein levels.
Tardif K., Hertig V., Dumais C., Villeneuve L., Perrault L., Tanguay J.F, & Calderone A. (2014). Nestin downregulation in rat vascular smooth muscle cells represents an early marker of vascular disease in experimental type I diabetes. Cardiovascular Diabetology, 13, 119.
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8

Western Blot Protein Analysis Protocol

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Cells were seeded overnight in 6 well plates and then treated for the designated period. Total cellular protein was extracted using RIPA buffer and the protein concentration was determined by using Pierce BCA protein assay kit (ThermoFisher, USA). Equal amounts of proteins were loaded onto a polyacrylamide gel and separated by SDS-PAGE. The proteins were transferred overnight to a nitrocellulose membrane (Biorad, USA). The membranes were blocked for 1h using membrane blocking solution (Life Technologies, USA) and then incubated overnight with primary antibody at 4°C. The primary antibodies used were rabbit monoclonal anti-Id1 (Calbioreagents, San Mateo, CA), rabbit monoclonal XIAP, rabbit monoclonal beta III tubulin (Tuj1), rabbit monoclonal anti-pSmad 1/5 (Cell Signaling Technology, MA, USA), rabbit polyclonal anti-Smad 1/5 (Upstate Biotechnology, NY, USA), rabbit anti-actin, rabbit polyclonal anti-GAPDH (Sigma, St. Louis, MO), and mouse monoclonal anti-spectrin (EMD Millipore, CA, USA). Rabbit polyclonal SOX2 (Abcam, USA), mouse monoclonal anti-NeuN (Chemicon, USA), and mouse monoclonal anti-Nestin (Chemicon, USA).
Kaye J., Mondal A., Foty R., Jia D, & Langenfeld J. (2022). Bone morphogenetic protein receptor inhibitors suppress the growth of glioblastoma cells. Molecular and cellular biochemistry, 477(5), 1583-1595.
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9

Neurosphere Differentiation Assay with sNgR-Fc

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The neurospheres described above were harvested, dissociated and cultured in medium supplemented with 500 ng/mL sNgR-Fc, but without epidermal growth factor or basic fibroblast growth factor to initiate and promote differentiation (sNgR-Fc group). For the control group, cells were differentiated without sNgR-Fc. After 7 days, NPC differentiation was assessed by immunocytochemistry. The following primary antibodies (neural stem cell-specific markers) were used: mouse monoclonal anti-nestin (1:100), rabbit monoclonal anti-glial fibrillary acidic protein (GFAP; 1:100; Chemicon, Hofheim, Germany), mouse monoclonal anti-RIP (1:50; kind gift from Dr Xu XM, University of Louisville School of Medicine, Louisville, Kentucky) and mouse monoclonal anti-β-tubulin III (1:100; Sigma-Aldrich, St. Louis, MO, USA). Incubations were performed at 4°C overnight. After washing with PBS, the sections were incubated with goat anti-rabbit IgG Alexa Fluor 594 and goat anti-Mouse IgG Alexa Fluor 594 (1:1000; Abcam, Cambridge, UK) at room temperature for 1 hour. The number of immunoreactive cells in each culture was calculated. Five visual fields (200×) were selected for each slide, and a total of 30 visual fields were counted for each parameter. The number of DAPI-stained cells was taken to represent the total number of cells. The percentage of immunoreactive cells was then calculated for each antibody.
He H.W., Zhang Y.L., Yu B.Q., Ye G., You W., So K.F, & Li X. (2019). Soluble Nogo receptor 1 fusion protein protects neural progenitor cells in rats with ischemic stroke. Neural Regeneration Research, 14(10), 1755-1764.
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10

Isolation and Differentiation of Rat NSCs

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Pregnant female Sprague-Dawley rats were provided by Experimental Animal Center, Xi’an Jiaotong University Health Science Center. All procedures involving animal work conformed to the ethical guidelines of the NIH Regulations for Experimentation on Laboratory Animals and set out by the Xi’an Jiaotong University.
NSCs were isolated from cerebral cortex of SD rat embryos on E14.5 days following the protocols of Gage FH and optimized in our laboratory [35 (link), 36 (link)]. Cells were cultured in serum free growth medium which contained DMEM/F12, 10 ng/ml bFGF, 20 ng/ml EGF, 1% penicillin, 1% streptomycin, 1% N2, and 2% B27 supplement (all from Invitrogen, Carlsbad, US) and 2.5 μg/ml heparin (Sigma, St. Louis, US). NSCs differentiation medium contained DMEM/F12 (1:1), 1% N2, 2% B27 supplement, 100 U/mL penicillin, 100 μg/mL streptomycin and 1% FBS. Primary cultured cells were sub-cultured every 5 days. NSCs identification was performed via immunocytochemistry staining. Primary antibodies, including monoclonal mouse anti-nestin (1:200, Millipore, Temecula, US), monoclonal mouse anti-β tubulin III (1:200, Millipore, Temecula, US) and polyclonal rabbit anti-GFAP (1:500, Santa Cruz, CA, USA) were used.
An J., Yan H., Li X., Tan R., Chen X., Zhang Z., Liu Y., Zhang P., Lu H, & Liu Y. (2017). The inhibiting effect of neural stem cells on proliferation and invasion of glioma cells. Oncotarget, 8(44), 76949-76960.
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