To analyze the surface maker of EPCs and TPCs, cells were seeded in glass‐bottomed dishes and cultured overnight. Then, the cells were fixed with 4% PFA, blocked with 5% BSA and incubated with primary antibodies overnight at 4℃. Then, the cells were incubated with secondary antibodies at room temperature in the dark for 1 h followed by DAPI (Sigma) staining. For tissue immunofluorescence staining, tumour tissues were removed, fixed with 4% PFA overnight, dehydrated in 30% sucrose/PBS solution and then embedded in OCT (Sakura). The 8‐μm‐thick cryosections were blocked, permeabilized, and then incubated with anti‐CD31 (AF3628, R&D Systems), anti‐PDGFRβ (CD140b) (14‐1402‐82, eBioscience), anti‐NG2 (AB5320, Chemicon), anti‐phospho‐Histone H3 (Ser10) (3458, Cell Signaling Technology), or anti‐α‐SMA‐Alexa Fluor 488 (34105, Cell Signaling Technology) antibodies overnight at 4℃, followed by incubation with corresponding secondary antibodies (Invitrogen) at room temperature in the dark for 1 h. DAPI was used for nuclear staining. The images were photographed under a Zeiss LSM 800 confocal microscope (Zeiss).
Anti phospho histone h3 ser10
Manufactured by Cell Signaling Technology
Sourced in United States, Germany
Anti-phospho-histone H3 (Ser10) is an antibody that specifically recognizes histone H3 phosphorylated at serine 10. This antibody can be used to detect mitotic cells and monitor cell division.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (6 mentions)
Anti histone h3, by Cell Signaling Technology (5 mentions)
Anti cleaved caspase 3 asp175, by Cell Signaling Technology (3 mentions)
Anti caspase 3, by Cell Signaling Technology (3 mentions)
Anti cyclin b1, by Cell Signaling Technology (3 mentions)
Anti flag, by Merck Group (3 mentions)
Anti cyclin b1, by Santa Cruz Biotechnology (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Anti phospho cdc2 tyr15, by Cell Signaling Technology (2 mentions)
Anti nfbd1, by Abcam (2 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Anti yap, by Cell Signaling Technology (2 mentions)
Anti pcna, by Santa Cruz Biotechnology (2 mentions)
Anti nestin, by Merck Group (2 mentions)
Mab1637, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Anti cd31 af3628, by R&D Systems (1 mentions)
Lsm800 confocal microscope, by Zeiss (1 mentions)
33 protocols using anti phospho histone h3 ser10
1
Immunofluorescence Staining of EPCs and TPCs
2
Mitochondrial Dynamics and Cell Cycle Analysis
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MitoTracker Green FM, DAPI, and Alexa Fluor 488– phalloidin were purchased from Life Technologies (Carlsbad, CA). Digitonin and etoposide were from Wako Pure Chemical Industries (Osaka, Japan). Mdivi-1 was from Enzo Life Sciences (Farmingdale, NY). Z-VAD-FMK was purchased from Peptide Institute (Osaka, Japan). NU6140 and BI2536 were from Santa Cruz Biotechnology (Santa Cruz, CA). The following antibodies were used for Western blotting and immunostaining: anti-Drp1, anti–cytochrome c (BD Biosciences, Billerica, MA), anti–γ-tubulin (Sigma-Aldrich, St. Louis, MO), anti-Smac (ProSci, Poway, CA), anti–phospho-Plk1 (Thr-210), anti-Plk1 (Abcam, Cambridge, United Kingdom), anti–phospho-CDK2 (Thr-160), anti-γ-H2AX, anti–phospho-histone H3 (Ser-10; Cell Signaling Technology, Beverly, MA), Alexa Fluor 488 anti-mouse immunoglobulin G (Life Technologies), anti–voltage-dependent anion-selective channel protein 1 (VDAC1), anti–glyceraldehyde 3-phosphate dehydrogenase (GAPDH), anti-CDK2, anti–cyclin A, anti–cyclin E, anti–cyclin B1, anti-actin, and horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology). The Western Lightning Plus-ECL chemiluminescence detection kit was purchased from PerkinElmer-Cetus (Boston, MA).
3
Analyzing Apoptosis and Cell Cycle by Flow Cytometry
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For apoptosis analysis by annexin V staining, total cell populations were collected by trypsinization and stained with annexin-V-FITC (1:20) and propidium iodide as per manufacturer’s instructions (Immune Quality Products). Cells were then analysed on a LSR-II (Becton Dickinson) cytometer using FACSDiva software (Becton Dickinson). For cell cycle analysis, BrdU and phospho-HistoneH3 analysis, cells were fixed in ice-cold 70% ethanol or methanol for at least 6 h and were then immunostained with an Alexa-488-conjugated antibody targeting BrdU (MoBU1, #B35130, 1:200), or anti-phospho-histone-H3 (Ser10, Cell Signaling, #9701, 1:300) in combination with Alexa-488-conjugated secondary antibodies (1:300). DNA staining was performed using propidium iodide in the presence of RNAse. At least 10,000 events per sample were analysed on a FACScalibur (Becton Dickinson). Data was analysed using FlowJo software.
4
Analyzing DNA Damage Response Proteins
5
Immunostaining of Drosophila Imaginal Discs
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Fixation and staining of Drosophila imaginal discs was performed in ‘watch-glass’ containers. Wing discs were fixed in 4% paraformaldehyde (PFA) for 20 min, then washed twice in PBS for 20 min. A blocking step was carried out for 2 h at 25°C using 5% fetal calf serum in PBST (PBS+0.1% Triton X-100). Discs were incubated with Anti-β-Galactosidase (1:1000, Promega, Madison, WI, USA) and Anti-phospho-Histone H3 Ser10 (1:500, Cell Signaling Technologies, Danvers, MA, USA) primary antibodies overnight at 4°C (Hendzel et al., 1997 (link); Wang et al., 2010 (link)). Four 20-min washes were performed with PBST before incubation with secondary antibody. Alexa Fluor® (1:500, Invitrogen) secondary antibody was incubated for 2 h at 25°C. We washed four times 20 min washes in PBST and then a final wash of 20 min in PBS to remove detergent. Wing discs were mounted on microscopy slides with VECTASHIELD® Mounting Medium (Vector Laboratories). Slides were kept dark at 4°C to reduce fluorophore fading.
6
Antibody Panel for Protein Expression Analysis
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The mouse monoclonal antibodies, namely anti-PR (sc-166169), anti-ERβ (sc-53494), anti-c-Jun (sc-74543), anti-p-c-Jun (sc-822), anti-JNK (sc-7345), anti-p-JNK (sc-6254), anti-C/EBPβ (sc-7962), and rat monoclonal antibody anti-ERα (sc-53493) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The rabbit polyclonal antibodies, including anti-phospho-cdc2 (Tyr15) (#9111), anti-cdc2 (#9112), anti-cleaved caspase 3 (Asp175) (#9661), anti-caspase 3 (#9662); rabbit monoclonal antibodies, including anti-phospho-cdc25C (Ser216) (#4901), anti-cdc25C (#4688), anti-phospho-histone H3 (Ser10) (#3377), anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) (#2118), anti-PR A/B (#3153), anti-FOXO1 (#2880), and anti-IGFBP1 (#31025) antibodies; and the anti-rabbit (#7074) and anti-mouse (#7076) IgG, horse radish peroxidase (HRP)-linked antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). The mouse monoclonal anti-Ki-67 (M7240) was purchased from Dako (Agilent; Santa Clara, CA, USA). All antibodies were used at the concentrations recommended by the manufacturers.
7
Lentiviral Knockdown of NFBD1 in DNA Repair
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The lentivirus-mediated shNFBD1 and shControl were purchased from Genechem, Shanghai, China. Hoechst 33342 were purchased from Beyotime Institute of Biotechnology (Nantong, China).The antibodies used in this study were anti-NFBD1 and RPA1 (Abcam, UK); anti-RAD51, anti-BRCA1 and anti-BRCA2 (Santa Cruz Biotechnology, USA); anti-γ-H2AX and anti-phospho-histone H3 (Ser10) (Cell Signaling Technology, Danvers, MA, USA).
8
Profiling PI3K and ALK Inhibitors
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The PI3K inhibitor copanlisib, TGX-221, alpelisib, idelalisib, and LY294002 were purchased from MedChemExpress (Princeton, New Jersey, USA). The PI3K inhibitor pictilisib, the ALK inhibitor crizotinib and NVP-TAE684 were obtained from ADOOQ (Irvine, California, USA). The antibodies against ALK, phospho-ERK1/2, Akt1/2, N-Myc, GAPDH, and beta-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA). Antibodies targeting Phospho-Akt (Ser473), Phospho-S6 Ribosomal Protein (Ser235/236), S6 Ribosomal Protein, and anti-Phospho-Histone H3 (Ser10) were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA). Other antibodies used in this study included anti-MAPK1/2 (Milipore, NG1946), anti-β-Tubulin antibody (Abcam, Ab6046).
9
Immunohistochemical Analysis of Colorectal Tissue
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Colorectal tissue sections were examined by a modified IHC method. Antigen retrieval was achieved by boiling tissue sections for 7 min in 0.01 mol/L sodium citrate, pH 6. Sections were blocked for 1 h in 1% BSA at room temperature and incubated in anti-PCNA (#13110, Cell Signaling Technology), anti-phospho-histone H3 (Ser10) (#53348, Cell Signaling Technology) or anti-γH2AX (ab2893, abcam) antibodies at 4°C overnight. After washing, samples were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG, and then by Biotin-XX-Tyramide amplification (Invitrogen), and streptavidin-HRP. Stained sections were visualized using 3,30-diaminebenzidine tetrahydrochloride (DAB) and counterstained with hematoxylin. IHC staining without primary antibody was used as a negative control.
10
Protein Expression and Cell Cycle Analysis
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Total protein was extracted from NE-4C cells using the NucleoSpin RNA/Protein Kit (Macherey-Nagel). Samples were separated by polyacrylamide gel electrophoresis (PAGE). Primary antibodies used for EP expression levels include rabbit polyclonal anti-EP1, −EP2, −EP3, −EP4 (1:200; Santa Cruz Biotechnology). Detection of rabbit monoclonal anti-Phospho-Histone H3 (Ser10) (1:1000; Cell Signaling) was used as a measure of cell splitting behaviour. Primary antibodies used for β-catenin expression levels were rabbit monoclonal anti-non-phospho (Active) β-catenin (Ser33/37/Thr41) and rabbit polyclonal anti-phospho-β-catenin (Ser552) (1:1000; Cell Signaling). Blots were reprobed with mouse monoclonal anti-β-Actin (1:10,000; Abcam). Visualization of bound anti-rabbit and anti-mouse horseradish peroxidise-conjugated secondary antibodies was achieved by incubation with ECL Prime Western Blotting Detection Reagent (GE Healthcare) and detection by Geliance 600 Imaging System (Perkin Elmer).
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