Immunocytochemistry was performed as we previously described39 (link). Briefly, cultured neurospheres or differentiated NPCs were washed with cold PBS (pH 7.4), and fixed with 4% paraformaldehyde (PFA) for 30 min. The cells were then washed with cold PBS containing 0.1% Tween for 4 times, 5 min each wash, and further blocked with 5% FBS for 1 h. Next the cells were incubated with the following primary antibodies: anti-Nestin (1:300, Abcam), anti-doublecortin (Dcx) (1:300, Abcam), mouse anti-BrdU (1:100; Roche Applied Science), mouse anti-β-tubulin III (Tuj-1, 1:500; Covance), rabbit anti-β-catenin (1:1000, Abcam), rabbit anti-glial fibrillary acidic protein (GFAP) (1:500; Invitrogen), mouse anti-PSA-NCAM (1:200, ThermoFisher). After PBS washing, the cells were then incubated with the primary antibodies listed above and with Cy3- or fluorescein isothiocyanate (FITC)-conjugated secondary antibodies. Vectashield (Vector Laboratory, Burlingame, CA) was used to coverslip the immunocytochemic slides. Immunostaining was analyzed using a fluorescence microscope (Olympus BX51). Quantification of neurite length in differentiated immature neurons (Tuj-1 positive) was performed using ImageJ-Neurite Tracer.
Anti nestin
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-Nestin is a primary antibody that detects the nestin protein, an intermediate filament protein expressed in neural stem and progenitor cells. It is commonly used in research applications to identify and study neural stem and progenitor cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Anti gfap, by Abcam (10 mentions)
Triton x 100, by Merck Group (8 mentions)
Anti sox2, by Abcam (6 mentions)
Anti tuj1, by Abcam (6 mentions)
Anti gfap, by Merck Group (6 mentions)
Anti akt, by Cell Signaling Technology (5 mentions)
Anti ki67, by Abcam (4 mentions)
Anti sox2, by Santa Cruz Biotechnology (4 mentions)
Anti β actin, by Merck Group (4 mentions)
Goat anti rabbit igg h l hrp, by Abcam (4 mentions)
Anti doublecortin dcx, by Abcam (3 mentions)
Anti β actin, by Santa Cruz Biotechnology (3 mentions)
Anti gap43, by Abcam (3 mentions)
Anti flag, by Merck Group (3 mentions)
Anti oct4, by Abcam (3 mentions)
Anti gapdh, by Cell Signaling Technology (3 mentions)
Anti sox2, by R&D Systems (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Anti olig2, by Merck Group (2 mentions)
66 protocols using anti nestin
1
Immunocytochemical Analysis of Neural Stem Cells
2
Quantifying Glioma and GSC Infection
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Immunofluorescence and quantification of infected glioma and GSC cultures was performed as previously published by our group [22] (link). Culturing GSC on laminin coated surfaces (rather than in spheres) has been shown to maintain their undifferentiated status and not alter their genome for up to 60 weeks [3] (link); therefore, cell samples were placed on laminin coated coverslips. Cells were probed with anti-IE1 (mAB810, Millipore), anti-Sox2 (Abcam), and anti-Nestin (Abcam) primary antibodies, then with AlexaFluor488 anti-mouse secondary antibody. Nuclei were counterstained with DAPI. IE1+ nuclei were counted with a fluorescence microscope fitted with a camera. Efficiency of infection was defined as the number of IE1+ cells divided by the total number of cells times 100.
3
Neural Stem Cell Immunostaining Protocol
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NSCs were washed with PBS and fixed in 4% paraformaldehyde (PFA) for 30 min at room temperature, followed by blocking with PBS (pH 7.4) containing 5% donkey serum, 0.1% Triton X-100, 0.02% sodium azide (NaN3), and 1% bovine serum albumin for 1 h at room temperature. This was followed by overnight incubation with primary antibodies at 4°C. Immunostaining was resumed by washing with PBS and incubating with secondary antibody (Invitrogen) for 1 h at room temperature followed by washing and then mounting in antifade fluorescence mounting medium (DAKO). The antibodies used in this study were anti-Ki-67, anti-TuJ1, and anti-nestin (Abcam, USA). Cell proliferation was measured with the Click-it EdU imaging kit (Invitrogen). Three independent experiments were repeated for each assay.
4
Histological Analysis of Mandibular Bone Regeneration
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After microCT scanning, specimens were processed for histological analysis. The mandibles were fixed with 4% paraformaldehyde at 4°C for 48 h, decalcified in 10% EDTA for 4 weeks, embedded in paraffin, and sliced in 5 μm sections along the axial plane. The slices were subjected to hematoxylin-eosin and immunohistochemical staining.
For hematoxylin-eosin staining, images were taken in 5 randomly selected high magnification fields (200×) per slide under a microscope. The new bone formation in the distraction gap was quantified with the ratio of trabecular bone volume/total volume using Image Pro-Plus analysis software (Media Cybernetics, Inc., Rockville, MD, U.S.) by an experienced pathologist.
For immunohistochemical staining, the tissue sections were incubated with anti-Nestin (1:100, Abcam, U.S.). The brown particles in the cytoplasm were considered positive. Images were photographed in 5 randomly selected fields (400×) per slide. The Nestin+ cells were counted manually by an experienced pathologist. All the experiments were conducted in triplicate.
For hematoxylin-eosin staining, images were taken in 5 randomly selected high magnification fields (200×) per slide under a microscope. The new bone formation in the distraction gap was quantified with the ratio of trabecular bone volume/total volume using Image Pro-Plus analysis software (Media Cybernetics, Inc., Rockville, MD, U.S.) by an experienced pathologist.
For immunohistochemical staining, the tissue sections were incubated with anti-Nestin (1:100, Abcam, U.S.). The brown particles in the cytoplasm were considered positive. Images were photographed in 5 randomly selected fields (400×) per slide. The Nestin+ cells were counted manually by an experienced pathologist. All the experiments were conducted in triplicate.
5
Quantitative Immunoblotting of Neural Markers
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Brain tissues containing the stab wounded region, or the SVZ were collected, and total proteins were extracted in a lysis buffer, containing 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 0.25 mM phenylmethylsulphonyl fluoride, 5 mg/ml aprotinin, and 1 mM sodium orthovanadate. After electrophoresed on 10% SDS-polyacrylamide gels, proteins were transferred onto nitrocellulose membranes, which were incubated appropriate primary antibodies overnight at 4°C: anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-GFAP (1: 2,000, Abcam), and Hes1 (1: 1,000, Abcam), anti-NDRG2 (1: 1,000, Abnova), anti-Nestin (1: 1,000, Abcam), or anti-activated Notch1 (NICD, 1: 1,000, Abcam). For detection, horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) and an enhanced chemiluminescence system (Amersham Biosciences, Piscataway NJ, USA) were used. For quantitation, the intensity of each band was quantified using Image-J software (NIH)., and then the data acquired were normalized to β-actin expression and further normalized to their corresponding controls.
6
Immunofluorescence Analysis of Neural Markers
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Immunofluorescence analysis was carried out essentially as previously described12 (link). Briefly, cells grown on poly-D-lysine-coated coverslips were fixed in 4% paraformaldehyde for 10 min at room temperature (RT), permeabilized with 0.3% Triton X-100/PBS, blocked with 3% BSA/PBS for 1 hr at RT, and incubated with anti-Tuj1 (1:1,000, Millipore), anti-GFAP (1:1,000, Millipore), anti-MAP2 (1:1,000, Millipore), anti-nestin (1:1,000, Abcam), or anti-CC3 (1:500, Millipore) antibody at 4 °C overnight, followed by an incubation with Alexa Fluor 488 or 555-conjugated goat anti-mouse or donkey anti-rabbit IgG (1:1,000, Invitrogen) with 0.1 μg/ml of 4′,6-diamidino-2-phenylindole (DAPI) for 1 hr at RT. Prolong Gold antifade reagent (Invitrogen) was used for mounting onto slides and immunofluorescence images were visualized with a Carl Zeiss AxioImager A2 microscope or Carl Zeiss Axiovert 200 M microscope equipped with a confocal laser scanning module LSM510.
7
Multimodal Immunofluorescence Analysis
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Cells were fixed for 10 min in 4 % paraformaldehyde, permeabilised and incubated with primary antibodies in 1× PBS with 10 % normal serum. Primary antibodies used were anti-Olig2 (1:500; Millipore), anti-GFAP (1:400; Millipore), anti-Ki67 (1:1,000; Abcam), anti-Nestin (1:1,000; Abcam), anti-Sox2 (1:200; Santa Cruz), anti-O4 (Sigma, 1:200), anti-Iba1 (1:200, Biocare), TuJ1 (1:1,000, Covance), anti-GFP (1:2,000, Abcam), anti-bromodeoxyuridine (BrdU) (1:250, Abcam) and anti-NFκB-p65 (1:500; Abcam). Primary antibodies were visualised using specific AlexaFluor secondary antibodies (Molecular Probes), and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Coverslips were mounted in Prolong Gold anti-fade mounting medium (Molecular Probes) and analysed using Zeiss AxioImager Z1 microscopes and AxioVision software.
8
Immunocytochemical Analysis of Hepatic Stellate Cells
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Nycodenz-isolated HSCs cultured for 1 day or for three passages were washed with PBS and fixed for 10 min with 4 % buffered formaldehyde (Merck, Darmstadt, Germany). Following permeabilization with 0.1 % Triton X-100 (in PBS containing 1 % bovine serum albumin), cells were incubated overnight with anti-αSMA (1/1000) (Sigma). Primary antibody binding was visualized using an Alexa488-labeled secondary antibody (1/200) (Invitrogen, Eugene, OR, USA). Images were taken with an AxioCam MRc5 digital camera (Carl Zeiss). NCAM1, nestin, and desmin stainings were performed as described previously [59 ]. Briefly, the cells were fixed, endogenous peroxidase was eliminated, and cells were permeabilized using PBS containing 1 % Triton X-100 (Sigma). Thereafter, the cells were blocked by 1-h incubation in PBS containing 1 % bovine serum albumin (Sigma), incubated with primary anti-NCAM1 (1/100) (Abcam), anti-Nestin (1/1000) (Abcam), or anti-Desmin (1/50) (Abcam) for 1 h, washed and incubated with secondary antibody (Envision, Dako) for 30 min. Detection was performed after 5 min incubation with liquid DAB and substrate chromogen (Dako). Counterstaining was performed using Mayer’s hematoxylin for 10 min. Preparations were then mounted for microscopic analysis (DMIL, Leica, Belgium).
9
Immunofluorescence Staining of Cell Cultures
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High (5 × 104 cells/cm2) and low (5 × 103 cells/cm2) density cell cultures were cultured for 4 days in basal media at 37 °C in 5 % CO2 in air before fixation in 70 % (v/v) ethanol for 15 min Fixed samples were permeabilised by incubation with 0.1 % Triton X-100 in PBS for 15 min before washing in PBS and blocking with pre-diluted goat serum (GeneTex, Irvine, CA, USA) for 30 min at 37 °C. Samples were then incubated with primary antibodies at dilutions between 1:50 and 1:250 overnight at 4 °C. Primary antibodies used were anti-cytokeratin 19 (FITC conjugated) (Santa Cruz Biotechnology, Dallas, TX, USA), anti-vimentin, anti-nestin and anti-fibronectin (all Abcam, Cambridge, UK). Non-conjugated primary antibodies were labelled with anti-rabbit IgG Alexa Fluor 488 (Abcam) for 30 min in the dark. Samples were then stained with DAPI for 15 min at room temperature to visualise nuclei before washing in PBS. Samples were then imaged using a Zeiss Axio Vert.A1 running Zen 2012 software (Carl Zeiss, Oberkochen, Germany).
10
Immunofluorescence Staining of Cultured Cells
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IF antibody assays were performed as previously described [8 (link)]. Cultured cells were placed on slides, treated with fixing solution I (4% paraformaldehyde plus 400 mM sucrose in PBS) and held at 37°C for 30 min. Slides were then treated with fixing solution II (fixing solution I plus 0.5% Triton X-100) and held at room temperature for 15 min. After washing with PBS, slides were treated with a blocking buffer (0.5% BSA in PBS) at room temperature for 1 h and washed three additional times with PBS prior to reacting with various primary antibodies (anti-Nestin [abcam] or anti-Tuj1 [GeneTex]) at 1:100 dilutions, either overnight at 4°C or for 1 h at 37°C. Slides were washed five times with cold PBS before reacting with FITC-conjugated anti-mouse IgG or TRITCconjugated anti-rabbit IgG (Sigma-Aldrich). After four more washes with cold PBS, slides were mounted and observed using a fluorescence microscope. DNA was stained with DAPI (Sigma- Aldrich) to localize nuclei.
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