For total protein extraction, cells were lysed in Nonidet-P40 lysis buffer (Boston Bioproducts, Ashland, MA) on ice for 30 min in the presence of a cocktail of protease and phosphatase inhibitors (Thermo Fisher Scientific) which included AEBSF, aprotinin, bestatin, E-64, leupeptin, and pepstatin A. Protein concentration was determined using the BCA assay (Pierce). Approximately 40 μg of total protein was loaded and separated on a precast 4–20% gradient polyacrylamide gel. After transfer to a PVDF membrane, proteins of interest were detected using relevant primary and HRP-conjugated secondary antibodies followed by chemiluminescent exposure (Super Signal West Pico ECL substrate, Pierce) on high performance chemiluminescence film (GE Healthcare, Little Chalfont, UK, Amersham Hyperfilm ECL). Primary antibodies used were anti-γ-tubulin (T3320, Sigma Aldrich), anti-decorin (H00001634-B01P, Abnova, Walnut, CA), anti-lumican (H00004060-D01P, Abnova), anti-serglycin (H00005552-M03, Abnova,), anti-glypican 5 (sc-84278, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Oct3/4 (611202, BD Biosciences, Franklin Lakes, NJ) and anti-nestin (611658, BD Biosciences).
Anti nestin
Manufactured by BD
Sourced in United States
Anti-Nestin is a laboratory reagent used for the detection and analysis of the cytoskeletal protein Nestin. It is a specific antibody that binds to Nestin, enabling its identification and quantification in various biological samples. The core function of Anti-Nestin is to provide a tool for researchers to study the expression and distribution of Nestin, which is often associated with stem and progenitor cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti sox2, by Abcam (5 mentions)
Anti gfap, by Merck Group (4 mentions)
Anti tuj1, by Fortrea (3 mentions)
Anti ki67, by Abcam (3 mentions)
Anti map2, by Merck Group (3 mentions)
Anti olig2, by Merck Group (2 mentions)
Anti gfap, by Agilent Technologies (2 mentions)
Anti map2, by Abcam (2 mentions)
Axio vert a1 imager a2, by Zeiss (2 mentions)
Anti neun, by Abcam (2 mentions)
Anti nanog, by Abcam (2 mentions)
Anti ki67, by Vector Laboratories (2 mentions)
Alexa fluorophore conjugated antibodies, by Thermo Fisher Scientific (2 mentions)
Hoechst 33258, by Thermo Fisher Scientific (2 mentions)
Anti gfap, by Cell Signaling Technology (2 mentions)
Anti mash1, by BD (2 mentions)
Anti sox2, by Santa Cruz Biotechnology (2 mentions)
Anti vimentin, by Merck Group (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Cocktail of protease and phosphatase inhibitors, by Thermo Fisher Scientific (1 mentions)
22 protocols using anti nestin
1
Protein Extraction and Immunoblotting Protocol
2
Immunofluorescence Staining of Neural Progenitors
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Neural progenitor cells grown on poly-L-ornithine/laminin coated 4-well chamber plates were fixed in 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.3% Triton-X-100 in Triton Buffer Solution (TBS) for 10 min, blocked with 2% FBS in TBS-0.05% Tween-20 for 20 min and stained with primary antibodies in blocking buffer at 4°C overnight. Primary antibodies included: anti-Nestin (611658; BD Pharmingen); anti-SOX2 (561469; BD Pharmingen); anti-RFP (600-401-379; Rockland); anti-ki67-Alexa fluor-488 (51-9007231; BD Stemflow Human neural Lineage analysis Kit); TuJ1 (MMS-435P, Covance); GFAP (ab7779, Abcam). The next day, the cells were stained with secondary antibodies (Alexa fluor 568 donkey anti-rabbit; Alexa fluor 488 donkey anti-mouse; Life Technologies, Inc.) in blocking buffer for 45 min and nuclei were counterstained with DAPI. Images were acquired using an Olympus FV1000 confocal microscope. Live culture images were also acquired using an Inverted Axioscope and AxioCam MRm (Carl Zeiss, Inc.). Image assembly was performed using Adobe Photoshop CS5 (Adobe Systems, Inc.).
3
Histological Evaluation of Glioma Samples
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Tumor samples fixed in 10% formalin were embedded in paraffin by the Duke Pathology Core and cut into 5-μm–thick sections using a Leica RM2235 microtome. Hematoxylin and eosin (H&E) staining was performed using standard protocols. Tumor grading was done using the following criteria: low-grade glioma (grade II) was indicated by an increased cellular density and the presence of Ki67 + cells; high-grade glioma (grades III and IV) was indicated by the presence of microvascular proliferation and/or the presence of pseudopalisading necrosis. Immunohistochemistry (IHC) was performed using an automated processor (Discovery XT, Ventana Medical Systems, Inc.). Antibodies used were anti-Olig2 (Millipore, #AB9610, 1:500), anti-GFAP (Dako, #Z0334, 1:2,000), anti-Nestin (BD Pharmingen, #556309, 1:200), anti-Ki67 (Abcam #ab16667, 1:200), anti-HA (Santa Cruz Biotechnology, #SC-805, 1:250), and anti–Tri-Methyl-Histone H3 Lys27 (Cell Signaling, #C36B11, 1:200). For rabbit antibodies, 10% normal goat serum in 2% BSA was used for the option/blocking step, and biotinylated goat anti-rabbit IgG (Vector Laboratories, #BA-1000, 1:300) was used for detection. For mouse antibodies, the Mouse on Mouse Basic Kit (Mouse on Mouse, Vector Laboratories, #BMK-2202) was used as directed for the option/blocking and detection steps.
4
Immunocytochemical Characterization of Cell Cultures
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Cells treated as described were fixed with precooled paraformaldehyde (4%, w/v) for 20 min, then permeabilized with 0.2% Triton X-100 for 20 min, blocked with 10% normal goat serum, and finally incubated overnight at 4 °C with the following primary antibodies: anti-MAP2 (1:500, rabbit IgG; Abcam, USA), anti-NeuN (1:800, mouse IgG; Abcam, USA), anti-Nestin (1:500, mouse IgG1; BD Biosciences, USA), anti-SOX2 (1:500, rabbit IgG; Abcam, USA), and/or anti-cleaved caspase-3 (1:1000, rabbit IgG; Cell Signal Technology, USA). The following day, the cells were treated with secondary antibody at room temperature for 1 h and the nuclei were counterstained for 10 min with DAPI. Immunoreactivity was visualized using a fluorescence microscope (AXIO Vert.A1&Imager A2, Carl Zeiss Microscopy GmbH, Germany).
5
Immunocytochemistry of Neural Markers
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Cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for 15 min at room temperature. After rinsing with PBS, cells were incubated with blocking/permeabilizing solution, which consists of PBS with 10% donkey serum and 0.1% Triton X-100 for 1 hr at room temperature. Subsequently, cells were incubated with primary antibodies overnight at 4°C. Primary antibodies used in this study were anti-nestin (1:1000; BD Sciences, USA) and anti-beta-III tubulin (1:500; Covance). In order to visualize the primary antibodies, cells were incubated with fluorescent-labeled secondary antibodies (Alexa 488-Alexa 594-, or Alexa 647-labeled IgG; Life Technologies) for 1 hr at room temperature. Images of stained cells were captured using an Olympus IX-70 inverted microscope, equipped with an Orca-ER CCD digital camera (Hamamatsu) and OpenLab software (Improvision). ImageJ (Wayne Rasband National Institute of Health, USA) was used for image analysis. In addition, images were analyzed by CellProfiler (version 2.1.1; cellprofiler.org ) for measuring fluorescent intensity. Three independent fields from each time point were used with matching exposure.
6
Immunohistochemical Characterization of Neural Cells
7
Immunofluorescence Characterization of ASS1-HLSCs
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Indirect immunofluorescence of cells was performed as described previously [4 (link)]. Briefly, ASS1-HLSCs cultured on chamber slides (Nalge Nunc International, Rochester, NY, USA) were fixed with 4% paraformaldehyde and/or permeabilized (only for intracellular markers) with 0.1% Triton X-100 buffer. Cells were then labeled with the following monoclonal antibodies: anti-albumin (R&D Systems), anti-nestin (BD Biosciences Pharmingen), anti-α-fetoprotein (αFP; R&D Systems), anti-nanog (Abcam, Cambridge, MA), anti-Sox2 (Abcam), anti-vimentin (Sigma-Aldrich), anti-cytokeratin 8 (CK8), anti-CK19, anti-Von Willebrand factor (all from Sigma-Aldrich), and anti-ASS1 (Thermo Fisher Scientific). Alexa Fluor 488 anti-rabbit IgG and Texas Red anti-mouse IgG (Thermo Fisher Scientific) were used as secondary antibodies and Hoechst 33258 dye (Sigma-Aldrich) was applied for nuclear staining. Labeling of cells with only secondary antibodies or substitution with nonimmune rabbit, rat, or mouse IgGs served as controls. Slides were analyzed by confocal microscopy using a Zeiss LSM 5 Pascal Model Confocal Microscope (Carl Zeiss International, Jena, Germany).
8
Comprehensive Neuronal Cell Marker Profiling
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The following primary antibodies were used for indirect immunofluorescence staining: anti-Cux1 (sc-13024, Santa Cruz Biotechnology); anti-Ctip2 (ab18465, Abcam); anti- GFP (GFP-1010, Aves Labs); anti-Pax6 (PD022, MBL); anti-bromodeoxyuridine (BrdU; catalog #555627, BD Biosciences); anti-phospho-histone-H3 (Ser10; catalog #09-797, Millipore); anti-Rapgef2 (Wei et al., 2007 (link)); anti-Rapgef6 (Yoshikawa et al., 2007 (link)); anti-β-catenin (catalog #610153, BD Biosciences); anti-N-cadherin (catalog #610920, BD Biosciences); anti-E-cadherin (catalog #610181, BD Biosciences); anti-afadin (catalog #ab90809, Abcam); anti-ZO-1 (catalog #61-7300, Life Technologies); anti-nestin (catalog #556309, BD Biosciences); anti-Tbr1 (catalog #ab31940, Abcam); and anti-c-Myc (catalog #23941-54, Nacalai Tesque). To detect immunoreactive signals, the following secondary antibodies were used: Alexa Fluor 488-conjugated anti-chicken Ig Y (catalog #703-545-155, Jackson ImmunoResearch); Alexa Fluor 647-conjugated anti-rabbit IgG (catalog #A21244, Life Technologies); CF647-conjugated anti-mouse IgG (catalog #20281, Biotium); CF488A-conjugated anti-IgG (catalog #20302, #20015, #20027, Biotium); CF488A anti-mouse IgG (Biotium); and CF555-conjugated anti-IgG (catalog #20231, #20038, #20233, Biotium). An Alexa Fluor 647-conjugated anti-Tbr2 antibody (catalog #51-4875, eBioscience) was also used.
9
Comprehensive Immunolabeling of Neural Cells
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For immunohistochemical analysis, cells were fixed with 4% paraformaldehyde (PFA) in 120mM phosphate buffer (PBS), pH 7.4, permeabilized with 0.05% Triton X-100 in PBS, blocked with 10% goat serum in PBS and subjected to immunohistochemistry staining with primary and secondary antibodies diluted in the blocking solution.
For immunolabelling the following antibodies at indicated dilutions were used: anti-Nestin (1:400; BD Bioscience), anti-Sox1 (1:100; R&D Systems), anti-Sox2 (1:200; Abcam), anti-Pax6 (1:200; DSHB), anti-Ki67 (1:200; Vector Labs), anti-TuJ1 (1:400; Covance), anti-Map2 (1:200; Millipore), anti-DCX (1:200; Millipore), anti-GFAP (1:200; Millipore), anti-O4 (1:50, Sigma-Aldrich), anti-GABA (1:200, Abcam), anti-vGlut1 (1:200; Millipore), anti-TH (1:100, Millipore), anti-Synaptophysin (1:100; Millipore), anti-PSD95 (1:200; Invitrogen), anti-vimentin (1:5000; Abcam), anti-S100β (1:1000; Sigma-Aldrich), anti-aquaporin 4 (AQP4, 1:100; Santa Cruz Biotechnology) and anti-excitatory amino acid transporter 2 (EAAT2, 1:100; Santa Cruz Biotechnology), secondary Alexafluorophore-conjugated antibodies (1:1000, Invitrogen). DNA was stained using Hoechst 33258 (1:10000, Invitrogen). All cells expressing a particular marker were counted manually and normalized to the total number of cells.
For immunolabelling the following antibodies at indicated dilutions were used: anti-Nestin (1:400; BD Bioscience), anti-Sox1 (1:100; R&D Systems), anti-Sox2 (1:200; Abcam), anti-Pax6 (1:200; DSHB), anti-Ki67 (1:200; Vector Labs), anti-TuJ1 (1:400; Covance), anti-Map2 (1:200; Millipore), anti-DCX (1:200; Millipore), anti-GFAP (1:200; Millipore), anti-O4 (1:50, Sigma-Aldrich), anti-GABA (1:200, Abcam), anti-vGlut1 (1:200; Millipore), anti-TH (1:100, Millipore), anti-Synaptophysin (1:100; Millipore), anti-PSD95 (1:200; Invitrogen), anti-vimentin (1:5000; Abcam), anti-S100β (1:1000; Sigma-Aldrich), anti-aquaporin 4 (AQP4, 1:100; Santa Cruz Biotechnology) and anti-excitatory amino acid transporter 2 (EAAT2, 1:100; Santa Cruz Biotechnology), secondary Alexafluorophore-conjugated antibodies (1:1000, Invitrogen). DNA was stained using Hoechst 33258 (1:10000, Invitrogen). All cells expressing a particular marker were counted manually and normalized to the total number of cells.
10
Immunoblotting Analysis of Neural Stem Cell Markers
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Anti-Sox2 (goat polyclonal, 1/1,000 dilution, R&D systems), anti-Nestin (mouse monoclonal, 1/1,000 dilution, BD), anti-GFAP (mouse monoclonal, 1/1,000 dilution, ImmunO), anti-NICD (rabbit polyclonal, 1/1,000, Cell Signaling), anti-HES1 (rabbit polyclonal, 1/1,000 dilution, Millipore), anti-Tuj1 (mouse monoclonal, 1/1,000 dilution, Abcam), anti-Symplekin (clone 25, mouse monoclonal, 1:1,000 dilution, BD), and anti-β-Actin (clone C4, mouse monoclonal, 1/1,000 dilution, Santa Cruz Biotech), anti-FLAG (clone M2, mouse monoclonal, 1/2,000 dilution, Sigma-Aldrich; rabbit polyclonal, 1/1,000 dilution, Cell Signaling), anti-HA (clone 3F10, rat monoclonal, 1/1,000 dilution, Roche), and anti-GFP (B-2, mouse monoclonal, 1/1,000 dilution, Santa Cruz Biotech) antibodies were used through the all WB analysis. As a secondary antibody, horseradish peroxidase-conjugated anti-rabbit (1/5,000 dilution, Vector Laboratories), anti-mouse IgG (1/5,000 dilution, Vector Laboratories), and anti-rat IgG (1/5,000 dilution, Santa Cruz Biotech) were used [57 (link), 58 (link)].
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