IHC were performed to examine cell proliferation marker as Ki67, pHH3, PCNA, and pERK in tumor tissues. IHC were also performed to examine inflammatory cell marker CD56. After being processed for paraffin embedding, 5 μm sections of tissue samples were prepared. Sections were boiled in 10 mM sodium citrate buffer (PH 6.0) for 20 min, and incubated in 0.3% hydrogen peroxide for 20 min and then blocked with 5% BSA for 1 h. Then incubated anti-Ki67 (Cell Signaling Technology, IHC, 1:400), anti-Phospho-Histone H3(Cell Signaling Technology, IHC, 1:50), anti-PCNA (Cell Signaling Technology, IHC, 1:4000) and anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling Technology, IHC,1:400), anti-CD56(Cell Signaling Technology, IHC, 1:800) antibodies overnight at 4 °C, followed by biotinylated secondary antibodies and DAB detection.
Anti phospho histone h3
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
Anti-phospho-histone H3 is a laboratory product used to detect the phosphorylation of histone H3 protein. Histone H3 phosphorylation is a key epigenetic modification associated with various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti cleaved caspase 3, by Cell Signaling Technology (6 mentions)
Anti β actin, by Merck Group (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Anti cyclin b1, by Cell Signaling Technology (2 mentions)
Nocodazole, by Merck Group (2 mentions)
Anti mecp2, by Abcam (2 mentions)
Anti mcm2, by Santa Cruz Biotechnology (2 mentions)
Anti map2, by Abcam (2 mentions)
Anti mecp2, by Cell Signaling Technology (2 mentions)
Nimodipine, by Merck Group (2 mentions)
Colchicine, by Merck Group (2 mentions)
Anti ki67, by Agilent Technologies (2 mentions)
Anti nicd, by Cell Signaling Technology (2 mentions)
Anti neun, by Merck Group (2 mentions)
Anti nestin, by Aves Labs (2 mentions)
Anti phospho s421, by Merck Group (2 mentions)
Anti aurkb, by Merck Group (2 mentions)
Anti dll1, by Abcam (2 mentions)
Anit histone h3, by Active Motif (2 mentions)
32 protocols using anti phospho histone h3
1
Immunohistochemical Analysis of Tumor Cell Markers
2
Quantifying Apoptosis and Proliferation
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To determine apoptotic cell death, a TdT-mediated dUTP nick-end labeling (TUNEL) assay was carried out on 10 μm cryosections using an in situ cell death detection kit (Roche Applied Science, Penzberg, Germany) according to the manufacturer’s instructions. To evaluate cell proliferation, immunofluorescent staining with anti-phospho-histone H3 (1:100 dilution, Cell Signaling Technology, Danvers, MA, United States) was performed on cryosections. The nuclei positive for TUNEL or phospho-histone H3 were counted, and the percentage of total nuclei was calculated.
3
Evaluating DNA Damage and Repair
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Cells after 30 min of X-ray irradiation were washed two times with cold PBS and fixed with 4% paraformaldehyde and 0.1% Triton X-100 during 30 min at room temperature. Then, the cells were washed three times with cold PBS and incubated overnight at 4 °C after the addition of 50 μL/well of a 1:500 dilution of anti-phospho-histone H3 (Cell Signalling Technology, Hertfordshire, UK). After this, the antibody was removed, and the plates washed with PBS. Then, 4 μM of Hoechst 33342 was added, and the plates were scored using an imaging cytometer (Celigo, Nexcelom Bioscience, Manchester, UK). After SDS-PAGE in 12% Bis-Tris precast gels using MOPS-SDS as the running buffer, Western blot analysis was carried out using the primary anti-Mps1 antibody at 1:500 dilution and the secondary anti-rabbit IgG Alkaline Phosphatase (AP)-linked antibody at 1:1000 dilution following a standard protocol. The protein β-actin was used as the loading control and detected using an anti-β-actin antibody.
4
Immunoblotting Analysis of Kinase Signaling
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Protein samples (15 μg per lane) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, and immunoblotted with anti‐MKK7, anti‐MKK4, anti‐JNK, anti‐phospho‐JNK, anti‐c‐Jun, anti‐phospho‐c‐Jun (Ser73), anti‐phospho‐histone H3 (Cell Signaling Technology), anti‐glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) (Cell Signaling Technology) and anti‐β‐actin (Sigma) antibodies. All blots were developed using an enhanced chemiluminescence detection system (GE Healthcare, Chalfont St. Giles, UK).
5
Evaluating Cellular Senescence and Mitochondrial Function
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The materials and their manufacturers’ information used in this study are the following: Dulbecco’s modified Eagle medium (DMEM)/F12, Penicillin-Streptomycin (P/S), 0.25% trypsin-EDTA and 10% Fetal Bovine Serum (FBS) from Gibco BRL (Grand Island, NY, USA); dimethyl sulfoxide from Invitrogen (Carlsbad, CA, USA); Tween® 20 and ECLTM Western blotting detection reagent from Amersham Life Science (Arlington Heights, IL, USA); etoposide from Selleckchem.com (Houston, TX, USA); anti-β Actin from Sigma (St. Louis, MO, USA); anti-Phospho-Histone H3 and anti-Histone H3 from Cell Signaling Technology (MA, USA); anti-CDK2 and anti-CDK4 from Santa Cruz Biotechnology; anti-GFAP from EMD Millipore (MA, USA); senescence detection kit from Abcam; Agilent Seahorse XF Cell Mito Test Kit from Agilent Technology (CA, USA); Doxorubicin from Sigma; Alexa Fluor® 594 conjugated Escherichia coli (K-12 strain) BioParticles® from Thermo Fisher Scientific (MA, USA); Tetramethylrhodamine Methyl Ester (TMRM) from Thermo Fisher Scientific.
6
Western Blot Analysis of Cell Cycle Markers
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Cells after treatment were washed with cold PBS and lysed on ice for 10 min in cell lysis buffer (Beyotime, Shanghai, China) containing PMSF (FDbio-tech, Hangzhou, China) and protease inhibitor cocktail (Biotool, Huston, USA). Lysates collected and centrifuged at 12,000× g for 20 min. Protein concentrations quantified by BCA Protein Assay Kit (Thermo Fisher, MA, USA), and 20 µg total protein separated on SDS/PAGE gels and subsequently transferred to polyvinylidene difluoride membranes. The membranes were blocked in 5% nonfat milk at room temperature for 1 h. Afterward, the membranes were incubated overnight at 4 °C with primary antibodies for anti-cyclinD1, anti-cyclinE2, anti-cyclinB1, anti-CDK1, anti–phospho-histone H3 (Cell Signal Technology, MA, USA), and GAPDH (Abcam, MA, USA), and followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies (anti-mouse IgG, HRP-linked antibody and anti-rabbit IgG, and HRP-linked antibody) (Cell Signal Technology, MA, USA) for 2 h at room temperature. The hybridization signals detected using an Enhanced Chemiluminescence Detection kit (Fdbio-tech, Hangzhou, China).
7
Quantitative Western Blotting Analysis
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Western blotting was carried out as previously described, using LI-COR Odyssey (LI-COR Biosciences) (Liu and Secombe 2015 (link)). Antibodies used were anti-phospho-histone H3 (#9701, 1:1000; Cell Signaling Technology), anti-histone H3 (#39763 or #39163, 1:5000; Active Motif), anti-α-tubulin (1:5000; Developmental Studies Hybridoma Bank, University of Iowa). The rabbit polyclonal KDM5 antibody was raised to amino acids 1418–1760 and has been previously published (Secombe et al. 2007 (link)).
8
Comprehensive Antibody and Drug Panel for Neurobiological Research
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The following primary antibodies were used: Anti-MeCP2 (Cell Signaling, 3456, 1:2,000), Anti-MeCP2 (Abcam, ab50005, 1:2,000), Anti phospho-S421 (custom made by Covance, 1:2,000), anti-β-Actin (Sigma-Aldrich, a5441, 1:5,000), anti-AURKB (Millipore, 04-1036, 1:1,000; Cell Signalling, 3094, 1:1,000), anti-DLL1(Abcam, Ab84620, 1:1,000), anti-NICD(Cell Signaling, 4147, 1:1,000), anti-phospho-Histone H3 (Ser10)(Cell Signalling, 3377, 1:1,000), anit-Histone H3 (Active Motif, 39163, 1:10,000), anti-S100b (SWANT, 1:500), anti-NeuN (Millipore, MAB377, 1:1,000), anti-BrdU (Accurate Chemical & Scientific, H7786, 1:1,000), anti-Ki67 (Dako, M7248, 1:500), anti-Tuj1 (Progema, G7121, 1:1,000), anti-MAP2(Abcam, ab32454, 1:1,000), anti-GFAP (Dako, Z0334, 1:1,000), anti-Nestin (Aves labs, NES, 1:500), anti-SOX2 (Millipore, MAB4343, 1:1,000), anti-MCM2 (Santa Cruz, sc-9839, 1:1,000). DyLight 680/800 conjugated secondary antibodies (Thermo Fisher Scientific) were used for Western blot. Alexa Fluor conjugated secondary antibodies (Invitrogen) were used for immunofluorescence staining. The following drugs were used: DAPT(Sigma-Aldrich), nocodazole (Sigma-Aldrich), colchicine (Sigma-Aldrich), roscovitine (Calbiochem), nimodipine (Sigma-Aldrich), Myr-CaMK IINtide (Calbiochem), STO-609 (Tocris), Bay K8644 (Calbiochem), Hesperadin (Selleckchem)
9
Immunostaining Reagents and Antibodies
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The following reagents were purchased from the indicated sources: rabbit anti-actin (A2066), Sigma-Aldrich; mouse monoclonal anti-βIII tubulin (clone 5G8), Promega; mouse monoclonal anti-bromodeoxyuridine antibody (clone Bu2a), DakoCytomation; anti-phospho Histone H3, Cell Signaling; mouse anti-pan epithelial monoclonal antibody (MAB1631), rat monoclonal anti-hGBP-1 antibody (1B1; 1B1 antibody does not work on frozen sections), and mouse anti-human CD31 (CBL468) monoclonal antibody, Chemicon; rabbit polyclonal anti-human CD31 (ab28364) and mouse monoclonal (KP1) to CD68 (ab9555), Abcam; Recombinant human interferon gamma (hIFN-γ), PBL Biomedical Laboratories; paclitaxel, Calbiochem (cat# 580555).
10
Tissue Sectioning and Immunostaining for Microscopy
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