The 5‐ethynyl‐2′‐deoxyuridine (EdU, 10 µmol/L, Invitrogen) was added to the culture medium following OGD/R. After an additional 24 hours, the cells were stained with Nestin antibody (1:100; Abcam) and EdU click reaction (Invitrogen). Immunoreactive cells were visualized using fluorescence microscopy (Leica). Differentiation of NSCs was induced by withdrawal of EGF and bFGF following OGD/R. At day 7, the differentiation was evaluated with Tuj1 (1:100; Abcam) staining. Each experiment was repeated at least three times.
Nestin antibody
Manufactured by Abcam
Sourced in United Kingdom
Nestin antibody is a protein used for the detection and analysis of nestin, an intermediate filament protein expressed in neural stem and progenitor cells. This antibody can be used in various research applications, such as immunohistochemistry and Western blotting, to identify and study the distribution of nestin-positive cells.
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Lab products found in correlation
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Rhegf, by Thermo Fisher Scientific (2 mentions)
Rh bfgf, by Thermo Fisher Scientific (2 mentions)
Fluorescence microscopy, by Leica (1 mentions)
Map2 antibody, by Merck Group (1 mentions)
Microplate reader, by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
Trypsin, by Merck Group (1 mentions)
Neurofilament antibody, by Merck Group (1 mentions)
Cold centrifuge, by Merck Group (1 mentions)
β catenin antibody, by Santa Cruz Biotechnology (1 mentions)
Gsk3β antibody, by Santa Cruz Biotechnology (1 mentions)
Image reader las 4000, by Fujifilm (1 mentions)
Lysis buffer, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Enhanced chemi luminescence (ecl), by Santa Cruz Biotechnology (1 mentions)
Bca protein assay reagent, by Bio-Rad (1 mentions)
Nfm antibody, by BioLegend (1 mentions)
Whatman, by Cytiva (1 mentions)
5 protocols using nestin antibody
1
Tracking Neural Stem Cell Proliferation
2
Stem Cell Differentiation Assay
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The following drugs and reagents were used: lithium chloride (Alfa Aesar B21573, Haverhill, MA, USA), β-catenin antibody (Santa Cruz C2206, Dallas, TX, USA), GSK-3β antibody (Santa Cruz AB15328), Nestin antibody (Abcam ab6142, Cambridgeshire, UK), MAP2 antibody (Merck Millipore AB5622, Burlington, MA, USA), GFAP antibody (Merck Millipore MAB3402C3), neurofilament antibody (Merck Millipore AB15328, MA), DMEM/F12 (Thermo Fisher, Waltham, MA, USA), basic fibroblast growth factor (bFGF, PeproTech, Rocky Hill, NJ, USA), epidermal growth factor (EGF, PeproTech), 0.25% trypsin (Sigma Aldrich, St. Louis, MO, USA), 0.01 mol/L RNase (Sigma Aldrich), 0.5 mg/L propidium iodide staining solution (Boster Biology, Wuhan, China), 5% Chloral hydrate (China), and 4% paraformaldehyde (Boster Biology). The equipment included a cold centrifuge (Sigma Aldrich, USA), an inverted light microscope (Olympus IX70, Tokyo, Japan), a microplate reader (Thermo Fisher), an incubator that was set at a temperature of 37°C and regulated with 5% CO2 (Thermo Fisher), and flow cytometer (FACSCalibur 2, Becton Dickinson, Franklin Lakes, NJ, USA).
3
Western Blot Analysis of Neuronal Markers
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Proteins were extracted with a lysis buffer (Cell Signaling Technology, Danvers, MA, USA) and quantified using the bicinchonic acid protein (BCA) protein-assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). Proteins were resolved by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Whatman GmbH, Dassel, Germany). After blocking in Tris-buffered saline with 5% milk and 0.1% Tween-20, the membrane was incubated with the neurofilament medium (NFM) antibody (Biolegend, San Diego, CA, USA) or Nestin antibody (Abcam, Beverly, MA, USA). The mouse monoclonal primary antibody to β-actin (Sigma-Aldrich) was used as the reference. Signals were visualized using an enhanced chemiluminescence reagent (ECL; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in Image Reader LAS-4000 software (Fujifilm, Tokyo, Japan).
4
Isolation and Characterization of Glioma Stem Cells
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Human brain microvessel endothelial cells (hBMECs) were purchased from ScienCell Research Laboratories (San Diego, CA, USA). The hBMECs were maintained in endothelial cell medium (ECM; ScienCell Research Laboratories). Six patient-derived primary glioma stem cells from WHO grade II to IV (grade II: GSC205 and GSC207; grade III: GSC306 and GSC307; grade IV: GSC406 and GSC408) were isolated, and neurosphere cultures were obtained as previously described [23 (link)]. The detailed clinicopathological information is presented in Table S2 . Briefly, freshly resected glioma samples were dissociated into single cells and grown in serum-free DMEM/F12 with 2% B27, 20 ng/mL rh-bFGF, and rh-EGF (Gibco, Gaithersburg, MD, USA). The stem cell markers of GSCs were detected by immunofluorescence using anti-CD133 (Abcam Technology, Cambridge, UK) and nestin antibodies (Abcam). The immunofluorescence staining of glial fibrillary acidic protein (GFAP; Abcam) and β-III tubulin (Abcam) was used to evaluate the multi-lineage differentiation capacity of GSCs.
5
Isolation and Characterization of Patient-Derived Glioma Stem Cells
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Six patient-derived primary glioma stem cells from WHO grade II to IV (grade II: GSC201 and GSC203; grade III: GSC302 and GSC305; grade IV: GSC403 and GSC406) were isolated and neurosphere cultures were performed as previously described (33) . The detailed clinicopathological information is presented in Supplementary Table 2. In brief, freshly resected glioma samples were dissociated into single cells and cultivated in serum-free DMEM/F12 with 2% B27, 20 ng/mL rh-bFGF, and rh-EGF (Gibco, Gaithersburg, MD, USA). The stem cell markers of GSCs were detected by immuno uorescence staining of CD133 (Abcam Technology, Cambridge, UK) and nestin antibodies (Abcam), and the multi-lineage differentiation capacity of GSCs was detected by immuno uorescence staining of GFAP and βIII tubulin (Abcam).
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