The following antibodies were used for immunofluorescence and immunoblot: Anti-SET/TAF1 (KM1725, KM1712, and KM1720; Nagata et al., 1998 (link)), Anti-hSgo1 (Kitajima et al., 2005 (link)), hSgo2 (Kitajima et al., 2006 (link)), HA (sc-7329 and sc-805 from Santa Cruz Biotechnology; 3F10 from Roche Diagnostics), Aurora B (611082; Becton Dickinson), pAurora A (T288)/B (T232; 2914S; Cell Signaling), borealin (M147-3; MBL), PP2A-A (sc-6112; Santa Cruz Biotechnology), Hec1 (GTX70268; Gene Tex) and Hec1 phospho Ser 55 (GTX70017; Gene Tex), cyclin B1 (sc-245; Santa Cruz Biotechnology), phospho-histone H3 Ser 10 (06–570; Millipore), β-tubulin (T4026; Sigma), α-tubulin (T9026; Sigma), His (M136-3; MBL), and Cenp-C (PD030; MBL) antibodies. Anti-centromere antibody (ACA) was provided by Y. Takasaki (Juntendo University, Tokyo, Japan). For immunofluorescence, secondary antibodies (Invitrogen Alexa Fluor) were used at 1:400. For immunoblotting, secondary antibodies (Santa Cruz Biotechnology or Wako) were used at 1:2,000.
Phospho histone h3 ser10
Manufactured by Merck Group
Sourced in United States
Phospho-Histone H3 (Ser10) is a lab equipment product that detects the phosphorylation of histone H3 at serine 10. This modification is associated with chromosome condensation during cell division.
Automatically generated - may contain errors
Lab products found in correlation
Cleaved caspase 3, by Cell Signaling Technology (3 mentions)
α tubulin, by Merck Group (2 mentions)
Picrosirius red, by Abcam (2 mentions)
Anti claspin, by Thermo Fisher Scientific (2 mentions)
Anti orc2, by Thermo Fisher Scientific (2 mentions)
Anti atr, by Proteintech (2 mentions)
F4 80, by Abcam (2 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (2 mentions)
Anti mcm2, by Abcam (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Cyclin b1, by Santa Cruz Biotechnology (1 mentions)
β tubulin, by Merck Group (1 mentions)
Paxillin, by Abcam (1 mentions)
Vinculin, by Merck Group (1 mentions)
Sp8 upright confocal microscope, by Leica (1 mentions)
Gm130, by BD (1 mentions)
Triton x 100, by Merck Group (1 mentions)
β catenin, by Abcam (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
21 protocols using phospho histone h3 ser10
1
Antibody Characterization for Cell Biology
2
Picrosirius Red and Immunofluorescence Staining
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Picrosirius red (Abcam; Cat# ab245887) and immunofluorescence staining were performed by standard methods using 8 μm paraffin sections. Antibodies used for immunofluorescence staining were F4/80 (Abcam; Cat# ab6640; 1:100), Ly6g (Abcam; Cat# ab25377; 1:100), phospho-histone H3 Ser10 (Millipore; Cat# 06-570; 1:200), anti-Claspin (Thermofisher; Cat# PA5-102840; 1:100), anti-MCM2 (Abcam; Cat# ab4461; 1:100), anti-Orc2 (Thermofisher; (Cat# PA5-70227; 1:100), and anti-ATR (Proteintech; Cat# 19787-1-AP; 1:100).
Sections were blocked in 3% BSA-PBST, and antibodies were diluted using 1% BSA-PBST. Primary antibodies were incubated overnight at 4 °C. Secondary antibodies were incubated for 1 h at room temperature. Slides were stained with DAPI during washing and coverslips mounted with ProLong Diamond Antifade Mountant (Invitrogen; Cat# P36961).
Sections were blocked in 3% BSA-PBST, and antibodies were diluted using 1% BSA-PBST. Primary antibodies were incubated overnight at 4 °C. Secondary antibodies were incubated for 1 h at room temperature. Slides were stained with DAPI during washing and coverslips mounted with ProLong Diamond Antifade Mountant (Invitrogen; Cat# P36961).
3
Immunofluorescence Imaging of Fibroblasts
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Fibroblasts were grown on 24 well plates with glass coverslips in each well. After 4 h of BCM treatment, cells were fixed with 4% paraformaldehyde (PFA) for 10 min, permeabilized 10 min with 0.1% Triton X-100 (Sigma) and 30 min blocking with 0.1 M L-lysine monohydrochloride in PBS. Samples were incubated with Cx43 (Sigma C6219, 1:4000), ZO-1 (Invitrogen 339 100, 1:100), β-catenin (Abcam ab6302, 1:2000), α-tubulin (Sigma T6074, 1:1000), GM130 (BD transduction Laboratories 610 822, 1:200), phospho-Histone H3 (Ser10) (Milipore 09–797, 1:1000), vinculin (Sigma) and paxillin (Abcam) at room temperature for 1 h. Samples were washed with PBS, incubated with secondary antibodies for 1 h at room temperature, stained with DAPI and/or rhodamine-phalloidin, washed in PBS and mounted in Citifluor® (Glycerol/PBS solution, Citifluor Ltd). Immunofluorescence was imaged on a Leica SP8 upright confocal microscope. Optimal gain and offset were set and kept constant during image acquisition. Minimum of three regions were imaged of each biological replicate.
4
Picrosirius Red and Immunofluorescence Staining
5
Antibody Sources and Reagents for Cell Signaling Analysis
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Primary antibodies were obtained from the following sources: PLK1 (05-844), phospho-Histone H3 (Ser10) (05806) from Millipore; Cyclin B1 (GNS1), Cyclin A (B-8), CDC27 (AF 3.1), and p55CDC20 (H-175) from Santa Cruz; PARP (9542), cleaved PARP (9542), Caspase-3 (9668), and cleaved Caspase-3 (8610) from Cell Signaling; MCL-1 (ADI-AAP-240-D) from Enzo; Securin (ab3305) and BCL-XL pS62 (ab30655) from Abcam; and from Santa Cruz; BCL-2 (7) from BD Transduction. β-Actin (A2228-100UL) from Sigma-Aldrich served as loading control.
Secondary antibodies for western blot analysis against rabbit (NA934V) and mouse (NXA931) IgG were obtained from GE Healthcare. Secondary antibodies used for immunofluorescence staining were obtained from Dako (F0313). Reagents were purchased from the following sources: Paclitaxel (T7402) Sigma-Aldrich, BI6727 (BYT-ORB181049) Selleckchem, propidium iodide (440300250) Acros Organics, RNase A (1007885) Qiagen, PE Annexin V (556421) and 7AAD (21-68981E) BD Biosciences, proTAME (1–440) Boston Biochem.
Secondary antibodies for western blot analysis against rabbit (NA934V) and mouse (NXA931) IgG were obtained from GE Healthcare. Secondary antibodies used for immunofluorescence staining were obtained from Dako (F0313). Reagents were purchased from the following sources: Paclitaxel (T7402) Sigma-Aldrich, BI6727 (BYT-ORB181049) Selleckchem, propidium iodide (440300250) Acros Organics, RNase A (1007885) Qiagen, PE Annexin V (556421) and 7AAD (21-68981E) BD Biosciences, proTAME (1–440) Boston Biochem.
6
Immunostaining Embryonic Development
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The immunostaining pattern of embryos was observed under a Zeiss LSM 780 confocal microscope. Primary antibodies were prepared from mouse, including anti-Gfap (RG marker) at 1:200 (Millipore), anti-HuC/HuD (early neuron marker) at 1:500 (Invitrogen), and rabbit polyclonal primary antibody against Phospho-Histone H3 Ser10 (Millipore) at 1:200 dilution. Secondary antibodies were goat anti-rabbit or anti-mouse Cy3-conjugated fluorescence at 1:500 (Millipore). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) at 1:1000 (Sigma).
7
Tumor Growth and Brain Metastasis Modeling
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U87 cells were stably transfected with lentivirus shTFF3, shHIF-1α or NC. Cells (106) were injected subcutaneously just caudal to the forelimbs in 4- to 6-week-old BALB/c nu/nu mice (Charles River, Beijing, China). Tumor diameters were measured with calipers at 7-day intervals, and volumes were calculated from four mice per data point (mm3 = width2 × length /2). For the intracranial inoculation, mice were anesthetized and a Hamilton syringe was used to inject 5×105 stable transfected cells through a 3-mm hole to the right of bregma, at a depth of 2.5 mm. The surgical zone was flushed with sterile saline, and the hole sealed with bone wax, and the skin over the injection site sutured. All animals were monitored daily and sacrificed at the end of the experiment. Then the whole brain was collected and fixed in 4% paraformaldehyde and cryoprotected in 30% sucrose. The brain was cryosectioned sagittally and stained with anti-GFP (1:200; CST, 2956S, USA) and phospho-Histone H3 (Ser 10) (1:200, Millipore, 06-570, USA). The experimental protocol was approved by the Dalian Medical University Animal Care & Use Committee, and all animals were housed in the Laboratory Animal Center of Dalian Medical University.
8
Antibody Detection of Cell Cycle Markers
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Mouse anti-Rb antibodies from BD Pharmigen (cat# 558385), or Cell Signaling Technology (cat# 8516s, or 9308s) were used for detection of Rb phosphorylated at S780, or S807/811, respectively. Other antibodies used were: topoisomerase (topo) II α (Cell Signaling Technology, cat# 12286s), histone H3 (tri methyl K9) (Abcam cat# ab8898), phospho-Histone H3 (Ser10) (Millipore cat# 06-570), phospho-CTD (Abcam cat# 5095), Mcl-1 (Cell Signaling Technology cat# 5453), FOXM1 (Abcam cat# ab175798), Ki67 (Abcam cat# ab16667), β-galactosidase (Abcam cat# ab9361), goat anti-mouse IgG-Alexa Fluor® 488 conjugated antibody (Life Technologies cat# A11017), goat anti-rabbit IgG-Alexa Fluor® 488 conjugated antibody (Life Technologies cat# A11008), and goat anti-chicken IgY H&L-Alexa Fluor® 647 conjugated antibody (Abcam cat# ab150171). For TUNEL assays, Roche cat#11767291910, 11767305001 and 11966006001 were used, and an annexin V-FITC kit was used for flow cytometry (Miltenyi Biotec cat# 130-092-052).
9
Immunohistochemistry Antibody Panel
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Primary antibodies for Sox2 (rabbit; ab97959), Sox2 (mouse; ab79351), Ki67 (rabbit; ab16667), Tbr2 (rabbit; ab23345), LacZ (chick; ab9361), Histone H3 trimethyl K4 (H3K4me3) (rabbit; ab8580), and GFP (chick; ab13970) were from abcam; Pax6 (rabbit; PD022) was from MBL; phospho-Histone H3 (Ser10) (rabbit; 06-570) was from Millipore; Cleaved Caspase-3 (rabbit; 9664S) was from Cell Signaling Technology; Beta-III tubulin (Tuj1) (mouse; MAB1195) was from R&D systems; GAPDH (mouse;016-25523) was from Wako, Ube2c (rabbit; 12134-2-AP) and ASPM (rabbit; 26223-1-AP) were from Proteintech. Antibodies for NF-YA (YA1-3) and NF-YC (YC5-3) were described previously (7 (link)).
10
Immunohistochemical Analysis of Cellular Markers
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Tissues were fixed in 10% formalin for 2 d and embedded in paraffin. Sections 5 μm thick were processed for H&E staining, IHC, or IF. The sections were incubated with primary antibodies against BrdU (Millipore, NA61), biotin-BrdU (Invitrogen, 03-3940), cytokeratin 8 (Developmental Studies Hybridoma Bank, TROMA-I), cleaved Caspase-3 (Cell Signaling, 9661), phospho-Histone H3 (Ser10; Millipore, 06-570), phospho-Histone H3 (Ser28; Sigma, H9908), KI-67 (Leica, NCL-Ki67p; BD Biosciences, 550609), Cyclin A2 (Santa Cruz Biotechnology, sc-596), Cre (Covance, PRB-1061C), or GFP (Abcam, ab6556). For detection of BrdU, the sections were treated in 2 M HCl for 10 min at 37°C prior to incubation with BrdU antibodies. Biotinylated secondary (Vector Laboratories) or fluorescent-conjugated secondary (Alexa fluor 488 or Alexa fluor 594, Invitrogen) antibodies were used for detection. Hoechst 33342 (10 µg/mL) was used as a counterstain for IF.
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