To induce SCAP neurogenesis, we seeded 1×106 cells into each ultralow adsorption dish (Corning, USA) and cultured them for 9 days. The neuron induction medium was described in our previous work.56 (link) Half of the neuron induction medium was changed every 3 days. After neuron induction, dynamic changes in neuron-like cells were observed for 3 days, 6 days, and 9 days under a microscope. After 9 days of neuron induction, immunofluorescence staining was applied to detect the markers of neurogenesis as described in our previous work.56 (link) The primer antibodies were as follows: Nestin (Cat No. ab 6320, Abcam, UK), βIII-tubulin (Cat No. T3952, Sigma, USA). At the conclusion of the experiment (5 weeks post-SCI), animals were euthanized by transcardiac perfusion with 4% paraformaldehyde in 0.9% sodium chloride. The spinal cord tissues, which had been previously embedded, were then sectioned into 5 μm slices and subjected to immunofluorescence staining. Immunofluorescence staining was performed according to a previous study.28 (link) The primer antibodies were as follows: Nestin (Cat No. ab 6320, Abcam, UK), βIII-tubulin (Cat No. T3952, Sigma, USA), NEFM (Cat No. OMA1-06110, Invitrogen, USA), and h-mitochondria (Cat No. ab 92824, Abcam, UK).
Nestin
Manufactured by Abcam
Sourced in United States, United Kingdom, China, Germany
Nestin is a class VI intermediate filament protein that is expressed in various stem and progenitor cells. It is commonly used as a marker for neural stem cells and is involved in the regulation of cell structure and motility.
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Lab products found in correlation
Glial fibrillary acidic protein (gfap), by Abcam (12 mentions)
Cd133, by Abcam (10 mentions)
Glial fibrillary acidic protein (gfap), by Agilent Technologies (8 mentions)
C myc, by Abcam (5 mentions)
Lxr α receptor, by Abcam (5 mentions)
Ssea4, by Abcam (5 mentions)
Cd133, by Miltenyi Biotec (5 mentions)
βiii tubulin, by Abcam (5 mentions)
Neuronal nuclei (neun), by Abcam (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Vimentin, by Abcam (4 mentions)
Olig2, by Merck Group (4 mentions)
β actin, by Santa Cruz Biotechnology (4 mentions)
E cadherin, by Abcam (4 mentions)
Tyrosine hydroxylase, by Abcam (4 mentions)
Lxr β receptor, by GeneTex (4 mentions)
Normal goat serum (ngs), by Dingguo (4 mentions)
Confocal laser scanning microscope, by Olympus (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Lsm 780, by Zeiss (4 mentions)
112 protocols using nestin
1
Optimizing SCAP-Induced Neurogenesis
2
Immunofluorescence Analyses of Stem Cell Markers
3
Immunofluorescence Staining of Flavivirus Proteins
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Cells were fixed in 4% paraformaldehyde solution (PFA; Sigma-Aldrich, St. Louis, MO, USA) in phosphate-buffered saline (Thermo Fisher Scientific), permeabilized with PBS/0.1% Triton X-100 (Sigma-Aldrich) and blocked in 4% bovine serum albumin (BSA, Sigma-Aldrich) in PBS. Then, cells were incubated with the primary antibodies diluted in PBS with 4% BSA. Primary antibodies specific for Flavivirus envelope protein E (clone 4G2, mouse, 1:500, Merck Millipore, USA), PAX6 (rabbit, 1:100, Sigma-Aldrich), Nestin (mouse, 1:100, Abcam, Cambridge, UK) were used. Cells were incubated overnight at 4 °C. The secondary antibodies used included the anti-mouse IgG Alexa Fluor-488 (goat, 1:250, Thermo Fisher Scientific) and the anti-rabbit IgG Alexa Fluor-546 (goat, 1:250, Thermo Fisher Scientific). DRAQ5 fluorescent probe solution (Thermo Fisher Scientific) was used to stain nuclei. Immunofluorescence was visualized by a confocal microscope (Leica, Wetzlar, Germany) under 63× magnification.
4
Neural Differentiation of DFCs and SHEDs
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A total of 1×10 5 DFCs and SHEDs were seeded into each well of a 6-well plate. Upon reaching 70% confluence, cells were cultured in neurogenic medium containing 2% dimethyl sulfoxide, 200 μM butylated hydroxyanisole (Sigma, USA), 25 mM KCl (Kelong, China), 2 mM valproic acid (Sigma, USA), 10 μM forskolin (Sigma, USA), 1 μM hydroxycortisone (Sigma, USA), and 5 μg/mL insulin (Gibco, USA) 3 (link). Cells grown in α-MEM supplemented with 10% FBS served as the negative control. After 4 h, cells were analyzed using immunofluorescence staining to determine the expression of the neural cell marker nestin (Abcam, USA). Images were captured and staining was analyzed under a fluorescence microscope (OLYMPUS, Japan). The expression of neurogenic genes GFAP, nestin and βIII-tubulin was analyzed using real-time PCR. The primer sequences are listed in Table 1 . Relative expression levels were calculated using the 2-ΔΔCT method and normalized to the reference GAPDH gene. The experiment was repeated three times.
5
Comprehensive Antibody Panel for Cellular Analysis
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SOX2 (Cat #3579), β-catenin (Cat #9562S & 9582S), FLAG (Cat # 9146S), ϒH2AX (Cat #9712S), H2AX (Cat #2595S) and K-48 (Cat # 8081) were purchased from Cell Signaling, Danvers, MA, USA. C-MYC (Cat # ab32072), Nestin (Cat # ab27952), Ki67 (Cat # ab16667) and Cyclin D1 (Cat # ab16663) were purchased from Abcam, Cambridge, MA, USA. Three individual antibodies to FBXO16 with Cat # NBP1–57614, AV53227 and PA566195 were procured from Novus Biologicals; Littleton, CO, USA, Sigma; St. Louis, Missouri, United States and Pierce; Waltham, MA, USA. The other antibodies used were Ub (Cat # Sc8017, Santa Cruz, Paso Robles, CA; USA), tubulin (Cat # T9026, Sigma) and actin (Cat # 691002, MP Biomedicals, CA, USA).
6
Immunofluorescence Staining of Neural Stem Cells
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Cells grown on coverslips were fixed in 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.3% Triton X-100 in PBS for 10 min, and blocked in 3% normal donkey serum in PBS for 2 h at room temperature. Primary antibodies were diluted in 3% normal donkey serum in PBS and applied at 4 °C overnight. The primary antibodies used in these experiments were as follow: Nestin (Abcam; 1:3000), Sox2 (Santa Cruz; 1:500), after rinsing in PBS three times and incubating for 2 h with CF488 and CF543 (Biotium; 1:1000), coverslips were washed three times, cell nuclei were stained with DAPI. Images were acquired on a Leica TCS SP2 confocal fluorescence microscope.
7
Immunofluorescence Analysis of Differentiated Cells
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Cells were cultured for 4 d on the stiff or soft gel-coated slides, and fixed with 4% paraformal- dehyde. After blocking in blocking buffer (containing 3% goat serum, 10% horse serum, 0.3% Triton X-100 in PBS) for 30 min at room temperature, the slides were incubated with primary antibodies against Sox9 (1:100; Abcam), Aggrecan (1:50; Abcam), ColII (1:50; Abcam), Runx2 (1:100), ColI (1;100), MAP2 (1:500; Abcam) , Nestin (1:100; Abcam) or NFL (1:500; Abcam), then with anti-mouse IgG conjugated with FITC (1:4,000; Abcam), anti-rabbit IgG conjugated with TRITC (1:4,000; Abcam) or anti-goat IgG conjugated with TRITC (1:4,000; Abcam). Finally, cell nuclei were visualized with DAPI (Sigma-Aldrich, St. Louis, Missouri, USA) and viewed under a Leica TCS SP5 confocal microscopy system (Leica, Germany).
8
Stem Cell Marker Expression Analysis
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To evaluate stem cell marker expression, neurospheres were dissociated mechanically or enzymatically with Accutase (Gemini Bioscience, Sacramento, CA). To facilitate adherence, cells were plated on poly-L-lysine/laminin coated four-well plates in neurosphere media. Cells were fixed in 4% paraformaldehyde, blocked and permeabilized with a 5% bovine serum albumin (BSA) with 0.6% Triton-× 100 and then treated with the primary antibodies Nestin (Abcam, Cambridge, MA), Sox2, Musashi 1, CD44, Bmi-1 (Cell Signaling Technology, Danvers, MA), CD133 (Biorbyt, Cambridge, UK) and A2B5 (A2B5 clone 105, ATCC, Manassas, VA). A “no primary control” was included for all antibodies tested for all cell lines. For these, the cells were incubated with only the antibody diluent (2.5% BSA, 0.3% triton, balance PBS). Cells were then treated with a fluorochrome-conjugated secondary antibody followed by Prolong Gold Antifade Reagent with DAPI (Thermo Fisher Scientific, Waltham, MA). Samples were examined under an EVOS FLoid Cell Imaging Station fluorescent microscope (Thermo Fisher Scientific, Waltham, MA).
9
Protein Isolation and Western Blot Analysis
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Total protein from the tissue or cell samples was isolated using RIPA lysis buffer (Beyotime Biotechnology). Protein concentrations were determined using the BCA protein assay kit (Beyotime Biotechnology). The remaining steps were performed according to previously described methods [
25 (link),
26 (link)] . The primary antibodies were as follows: GSK-3β (1:1000; CST, Boston, USA), β-catenin (1:1000; CST), nestin (1:1000; Abcam, Cambridge, UK), cyclin D1 (1:1000; CST), β-actin (1:10,000; Proteintech, Wuhan, China), and histone 3 (1:1000; Abmart, Shanghai, China). ImageJ (NIH, Bethesda, USA) was used for quantification.
25 (link),
26 (link)] . The primary antibodies were as follows: GSK-3β (1:1000; CST, Boston, USA), β-catenin (1:1000; CST), nestin (1:1000; Abcam, Cambridge, UK), cyclin D1 (1:1000; CST), β-actin (1:10,000; Proteintech, Wuhan, China), and histone 3 (1:1000; Abmart, Shanghai, China). ImageJ (NIH, Bethesda, USA) was used for quantification.
10
Immunostaining of Neuronal Cell Markers
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Cells were fixed with 4% paraformaldehyde and blocked with 5% bovine serum albumin in PBS. Cells were stained with primary antibodies overnight at 4°C. Dilutions were as follows: Nestin, 1:100 (Abcam, Cambridge, UK); TH, 1:200 (Millipore, CA, USA); β III tubulin (Tuj1; Millipore, CA, USA), 1:200 (Millipore, CA, USA); LXR α receptor, 1:200 (Abcam, Cambridge, UK); LXR β receptor, 1:200 (Abcam, Cambridge, UK). Appropriate secondary antibodies anti-rabbit IgG-dylight 549(Abbkine, CA, USA) and anti-mouse IgG-dylight 488(Abbkine, CA, USA); nuclear stain 4, 6-diamidino-2-phenylindole (DAPI; Beyotime Biotechnology, Shanghai, China); and cytoskeletal stain Texas Red phalloidin (F-actin; Molecular Probes, Eugene, OR, http://probes.invitrogen.com ) were used for detection and visualization [7 (link)]. Finally, the numbers of total cells and TH/Tuj1 positive cells were determined using a laser scanning confocal microscope (Nikon A1*R, Japan) and fluorescence intensity was analysis by NIS-Elements AR 4.2 (Nikon, Japan).
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