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Anti phospho histone h3 ser10 antibody

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The Anti-phospho-Histone H3 (Ser10) antibody is a laboratory reagent used to detect and quantify the phosphorylation of histone H3 at serine 10. This modification is associated with cell cycle progression and chromatin condensation during mitosis.

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Lab products found in correlation

Axio observer d1 microscope, by Zeiss (1 mentions) Alexa fluor 546 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions) Anti ha, by Santa Cruz Biotechnology (1 mentions) Anti sox9 antibody, by Merck Group (1 mentions) Hematoxylin qs, by Vector Laboratories (1 mentions) Proteinase k solution, by Nacalai Tesque (1 mentions) Hyaluronidase, by Merck Group (1 mentions) Eosin y, by Merck Group (1 mentions) Immpact dab peroxidase substrate, by Vector Laboratories (1 mentions) Vectastain elite abc kit, by Vector Laboratories (1 mentions) Methyl green nuclear counterstain, by Vector Laboratories (1 mentions) Facscan flow cytometer, by BD (1 mentions) Dig utp, by Roche (1 mentions) Hoechst 33342, by Thermo Fisher Scientific (1 mentions) Tcs sp8 x confocal microscope, by Leica (1 mentions) Alexa fluor 647 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions) Aqua poly mount, by Polysciences (1 mentions) Cy3 conjugated goat anti rabbit, by Jackson ImmunoResearch (1 mentions) Alexa 488 conjugated donkey anti rabbit, by Jackson ImmunoResearch (1 mentions) Donkey anti rabbit cy3 secondary antibody, by Jackson ImmunoResearch (1 mentions)

Spelling variants of this product

Anti-phospho-histone H3 (52 citations) Phospho-histone H3 (40 citations) Anti-phospho-histone H3 (Ser10) (32 citations) Phospho-Histone H3 (Ser10) (21 citations) Anti-histone H3 antibody (11 citations) Anti-phospho-Histone H3 antibody (6 citations) Phospho-Histone H3 antibody (5 citations) Phospho-H3 (Ser10) (4 citations) Histone H3 antibody (4 citations) Anti-H3 phospho-Ser10 (2 citations)

11 protocols using anti phospho histone h3 ser10 antibody

1

Immunohistochemical Profiling of Cardiac Development

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Immunohistochemical studies were performed as described previously8 (link),17 . The GATA6 antibody18 (link) was used at 1/1000 dilution. Goat polyclonal IgG GATA4 (C20) antibody was purchased from SANTA CRUZ (SC-1237X; dilution 1/600). The following antibodies were purchased from ABCAM: Semaphorin3C (ab 135842; dilution 1/350), SOX9 (ab3697, dilution 1/100), Periostin (POSTN) (ab14041, dilution 1/1000), alpha smooth muscle actin (ab5694, dilution 1/750), total and cleaved Versican (ab19345 and ab177480, dilution 1/250 each), and NCID (ab8925, dilution 1/500). The Anti-phospho-Histone H3 (Ser10) antibody was from MILLIPORE (06-570, dilution 1/750). The biotinylated anti-Goat IgG and anti-Rabbit antibodies were from Vector Laboratories (BA5000) and Jackson (Cederlane) (711-065-152) respectively. Streptavidin-HRP conjugate was from Perkin Elmer (NEL 750000 1EA). Immunofluorescence was carried out using Anti-HA (Santa Cruz, Santa Cruz, CA, USA, sc-805) and Alexa Fluor 546 Goat Anti-Rabbit IgG (Life Technologies, Carlsbad, CA, USA, A-11035) at a dilution of 1/500 respectively. Image acquisition was completed using the Zeiss AxioObserver D1 microscope (Oberkochen, Germany).
Gharibeh L., Komati H., Bossé Y., Boodhwani M., Heydarpour M., Fortier M., Hassanzadeh R., Ngu J., Mathieu P., Body S, & Nemer M. (2018). GATA6 regulates aortic valve remodeling and its haploinsufficiency leads to RL-type Bicuspid Aortic Valve. Circulation, 138(10), 1025-1038.
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2

Immunohistochemical Analysis of Bone Development

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Bone samples were fixed in 4% paraformaldehyde, decalcified, and embedded in paraffin, as previously described [14 (link)]. To unmask antigens, tissue sections were boiled in 10 mM citrate buffer [for phospho-Histone H3, and Spp1 (Secreted phosphoprotein 1)], or incubated in 700 U/mL proteinase K solution (Nacalai Tesque) (for Sox9) at 37°C for 5 minutes, or treated with 1mg/mL hyaluronidase (Sigma-Aldrich) at 37°C (for Runx2). Immunohistochemistry was performed using the Vectastain Elite ABC kit (Vector) and Immpact DAB peroxidase substrate (Vector). Sections were counterstained with Hematoxylin QS (Vector) or Methyl Green Nuclear Counterstain (Vector). The following primary antibodies were used: anti-Spp1 antibody (RB9097-PO; Thermo Scientific), anti-Ihh antibody (sc-1196; Santa Cruz), anti-RUNX2/CBFA1 antibody (PA1224; Boster Biological), anti-HAND1 antibody (GTX11846; GeneTex), anti-SOX9 antibody (AB5535; Millipore), and anti-phospho-Histone H3 (Ser10) antibody (06–570; Millipore).
For alcian blue staining, sections were treated with 3% acetic acid, stained with 1.5 mg/mL alcian blue 8GX in 3% acetic acid solution, and then counterstained with Eosin Y (Sigma-Aldrich). For von Kossa staining, sections were incubated in 1% silver nitrate overnight under UV light, then incubated in 5% sodium thiosulfate, and counterstained with Eosin Y.
Laurie L.E., Kokubo H., Nakamura M., Saga Y, & Funato N. (2016). The Transcription Factor Hand1 Is Involved In Runx2-Ihh-Regulated Endochondral Ossification. PLoS ONE, 11(2), e0150263.
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3

Cell Cycle Analysis by Flow Cytometry

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Cells were detached from dishes using trypsin, centrifuged, and fixed using 75% ethanol for at least 1 h. Cells were then washed 3 times in PBS, treated with 20 μg/mL RNase and 50 μg/mL propidium iodide (PI) at 37°C for 30 min, and analyzed on a FACScan flow cytometer (Becton Dickinson). For phospho-histone H3 (Ser10) detection, cells were washed and permeabilized using 0.5% triton X-100, incubated with anti-phospho-Histone H3 (Ser10) antibody (Millipore, 1:1000) for 1 h at room temperature and then secondary antibody before RNase treatment and PI staining.
Wen D., Wu J., Wang L, & Fu Z. (2017). SUMOylation Promotes Nuclear Import and Stabilization of Polo-Like Kinase 1 to Support its Mitotic Function. Cell reports, 21(8), 2147-2159.
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4

Immunohistochemical Visualization of Dividing Cells

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Immunohistochemistry protocols follow [70 (link)]. We visualized En using En4F11 (gift from N. Patel) and dividing cells using pH3 (anti-phospho-Histone H3 (Ser10) Antibody; Millipore) at 1 µg/mL. Specimens were counterstained with Hoechst, mounted in 80% glycerol supplemented with 0.2 M TRIS buffer and 0.024 M n-propyl gallate using clay feet on coverslips to prevent distortion, and photographed on a Nikon E600 Ellipse epifluorescence microscope and a Spot Insight QE digital camera (Diagnostic Instruments, Sterling Heights, MI, USA) and Spot Advanced software.
Constantinou S.J., Duan N., Nagy L.M., Chipman A.D, & Williams T.A. (2020). Elongation during segmentation shows axial variability, low mitotic rates, and synchronized cell cycle domains in the crustacean, Thamnocephalus platyurus. EvoDevo, 11, 1.
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5

Whole-mount in situ hybridization and immunohistochemistry

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WISH and IHC were performed as previously described (King and Newmark, 2013 (link); Pearson et al., 2009 (link); Reddien et al., 2005 (link)). Briefly, DIG-UTP- (Roche Diagnostics, Mannheim, Germany) labeled sense and antisense riboprobes were allowed to hybridize at 56°C for 20-40 h. Hybridized riboprobes were either detected via NBT/BCIP precipitation or fluorescence, using Anti-Digoxigenin-POD Fab fragments (1:100; Roche) anti-phospho-histone H3 (Ser10) antibody (1:1000, Millipore, Merck Chemicals, Darmstadt, Germany) and Alexa Fluor 647-conjugated goat anti-rabbit IgG (1:1000; Life Technologies, Darmstadt, Germany) were used as primary and secondary antibodies to detect cells in G2-to-M phase. Hoechst 33342 (5 µg/ml; Thermo Fisher Scientific) was used for nuclear counterstaining. Samples were embedded in Aqua Poly/Mount (Polysciences, Hirschberg an der Bergstraße, Germany) on a glass slide and imaged using a Leica, TCS SP8X confocal microscope. Cell counting was normalized to the body surface of each specimen, as determined by ImageJ2 (Schindelin et al., 2015 (link)).
Van Roten A., Barakat A.Z., Wouters A., Tran T.A., Mouton S., Noben J.P., Gentile L, & Smeets K. (2018). A carcinogenic trigger to study the function of tumor suppressor genes in Schmidtea mediterranea. Disease Models & Mechanisms, 11(9), dmm032573.
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6

Immunostaining of C. elegans Gonads

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Dissected gonads stained with either the rabbit anti-phospho-histone H3 (Ser10) antibody (1:400 dilution; Millipore, Burlington, MA) or the rabbit anti-RME-2 antibody (1:50 dilution, kindly provided by Barth Grant) (Grant and Hirsh 1999 (link)) were fixed in 3% paraformaldehyde for 1 hr, as described (Rose et al. 1997 (link)). Dissected gonads stained with the rabbit anti-GLD-1 primary antibody (1:150 dilution; Jan et al. 1999 (link)) were fixed in 1% paraformaldehyde for 10 min with a 5-min postfix step in ice-cold methanol. Primary antibodies were detected using either Cy3-conjugated goat anti-rabbit or Alexa 488-conjugated donkey anti-rabbit secondary antibodies (1:500 dilutions; Jackson ImmunoResearch). For the strong loss-of-function lin-41(tn1541tn1618) mutant, gonads were dissected from postdauer animals on the first day of adulthood.
Spike C.A., Huelgas-Morales G., Tsukamoto T, & Greenstein D. (2018). Multiple Mechanisms Inactivate the LIN-41 RNA-Binding Protein To Ensure a Robust Oocyte-to-Embryo Transition in Caenorhabditis elegans. Genetics, 210(3), 1011-1037.
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7

Analyzing Myocardial Proliferation by Immunofluorescence

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HIF1α expression was analyzed by immunofluorescence in either wholemount or on wax sections using an anti-HIF1α antibody (NB100-449, Novus Biologicals) and a donkey anti-rabbit Cy3 secondary antibody (Jackson ImmunoResearch) as previously described (Geffers et al., 2007 (link)). Nuclei were counterstained with TO-PRO3 (Life Technologies). Images were captured on a LSM 710 confocal microscope (Carl Zeiss Microscopy). Myocardial proliferation was assessed by wholemount immunofluorescence using an anti-phospho-Histone H3 (Ser10) antibody (06-570, Merck Millipore) and a donkey anti-rabbit Cy3 antibody (Jackson ImmunoResearch). Nuclei were counterstained with DAPI. A z series of optical sections 1.5 μm apart encompassing the entire heart were captured. Every 20th optical section, a total of 4–7 sections per heart, was analyzed using ImageJ software (NIH) and the mean mitotic index calculated. This was expressed as the percentage of phospho-Histone H3 positive area per total myocardial area.
O'Reilly V.C., Floro K.L., Shi H., Chapman B.E., Preis J.I., James A.C., Chapman G., Harvey R.P., Johnson R.S., Grieve S.M., Sparrow D.B, & Dunwoodie S.L. (2014). Gene–environment interaction demonstrates the vulnerability of the embryonic heart. Developmental biology, 391(1), 99-110.
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8

Cell Proliferation and Mitosis Evaluation

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DNA synthesis was determined by measuring the incorporation of BrdU by using a fluorescence-conjugated antibody against BrdU (BD Biosciences, San Agustin de Guadalix, Spain), co-stained with PI, and analysed in a Coulter Epics-XL flow cytometer.
To analyse the mitotic fraction, fixed cells were incubated with the anti-phospho-Histone H3 (ser 10) antibody (Merck Millipore, Madrid, Spain) followed by Cy2-conjugated secondary antibody (Jackson ImmunoResearch, Newmarket, UK). Stained cells were then counterstained with PI and analysed for Cy2 and PI fluorescence in a Coulter Epics-XL flow cytometer.
Mansilla S., Vizcaíno C., Rodríguez-Sánchez M.A., Priebe W, & Portugal J. (2015). Autophagy modulates the effects of bis-anthracycline WP631 on p53-deficient prostate cancer cells. Journal of Cellular and Molecular Medicine, 19(4), 786-798.
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9

Phospho-histone H3 Immunostaining in Embryos

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Embryos were fixed overnight in 4% paraformaldehyde at
4ºC and permeabilized in acetone at −20ºC. Embryos were
incubated with Anti-phospho-histone H3 (Ser10) Antibody (EMD Millipore)
overnight and again with secondary antibody Donkey Anti-Rabbit IgG H&L
(Alexa Fluor® 488) (Abcam).
Nissim S., Weeks O., Talbot J.C., Hedgepeth J.W., Wucherpfennig J., Schatzman-Bone S., Swinburne I., Cortes M., Alexa K., Megason S., North T.E., Amacher S.L, & Goessling W. (2016). Iterative use of nuclear receptor Nr5a2 regulates multiple stages of liver and pancreas development. Developmental biology, 418(1), 108-123.
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10

Whole-Mount Staining of Mouse Hindpaw Tissues

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Mouse hind paw tissues were processed for whole-mount staining. Briefly, full-thickness paw skin was dissected and fixed in 4% paraformaldehyde (PFA) in PBS for 4–6 hours at room temperature, washed in PBS, and then blocked with 0.2% Triton X-100, 5% normal donkey serum, 1 % BSA in PBS overnight at 37°C. The samples were then incubated with primary antibodies for 48–72 h and secondary antibodies for 24 h on a rocker at 37°C. The anti-phospho-histone H3 (Ser10) antibody was from EMD Millipore (06–570) and was used at a 1:100-fold dilution; anti-Flk-1 (VEGFR2) antibody was from BD Biosciences (555307) and was used at 1:100-fold dilution; anti-ERG antibody was from Abcam (AB92513) and was used at 1:200-fold dilution; goat anti-rabbit IgG H&L (Alexa Fluor®) 568, goat anti-rat IgG H&L (Alexa Fluor®) 568, goat anti-Chicken IgY H&L (Alexa Fluor®) 488, and goat anti-rat IgG H&L (Alexa Fluor®) 488 was from ThermoFisher and was used at a 1:400-fold dilution. Tissue samples were mounted with Vectashield anti-fade mounting medium (Vector Laboratories) on individual slides and imaged on a LaVision TriM Scope II, as described in ‘In vivo imaging.’
Vowels J.J, & Payne G.S. (1998). A Role for the Lumenal Domain in Golgi Localization of the Saccharomyces cerevisiae Guanosine Diphosphatase. Molecular Biology of the Cell, 9(6), 1351-1365.
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