Skip to main content
SpringerLink
Log in

Immunostaining Analysis of Tissue Cultured Cells and Tissue Sections Using Phospho-Histone H3 (Serine 10) Antibody

  • Protocol
Chromatin Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1288))

Abstract

Post-translational modifications of histones play an important role in regulation of gene expression through condensation and decondensation of chromatin structure. These modifications include acetylation, methylation, phosphorylation and ubiquitination. Phosphorylation on histones is associated with cellular responses such as DNA damage, transcription, chromatin compaction and mitosis or meiosis. One of the most extensively studied modifications of histones is the Serine 10 phosphorylation on histone H3 N-terminal tail. This specific phosphorylation on Histone H3 has been associated with condensation and transcriptional inactivation of mitotic chromosomes, but recent studies have suggested a role for this specific phosphorylation in chromatin relaxation and activation of transcription in interphase cells. Co-immunostaining analysis of cells using antibodies specific to serine 10P-Histone H3 together with those to cell cycle specific markers will allow us to determine the nature of phosphorylation in a cell cycle-specific manner. In a complex system, such as tissue specimens, analysis using P-Histone H3 and a cell type specific antibody will allow identification of specific cells that are affected by this histone modification. This is of particular interest in the field of cancer biology or neurobiology where identification or quantification of the transcriptionally active or mitotic cells will enable one to evaluate the progression of the disease development. The protocol described here provides details on how co-immunostaining and analysis can be performed in tissue cultured cells or tissue sections.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Nowak SJ, Corces VG (2004) Phosphorylation of histone H3: a balancing act between chromosome condensation and transcriptional activation. Trends Genet 20(4):214–220

    Article  CAS  PubMed  Google Scholar 

  2. Mahadevan LC, Willis AC, Barratt MJ (1991) Rapid histone H3 phosphorylation in response to growth factors, phorbol esters, okadaic acid, and protein synthesis inhibitors. Cell 65(5):775–783

    Article  CAS  PubMed  Google Scholar 

  3. Chen R, Wierda WG, Chubb S, Hawtin RE, Fox JA, Keating MJ, Gandhi V, Plunkett W (2009) Mechanism of action of SNS-032, a novel cyclin dependent kinase inhibitor, in chronic lymphocytic leukemia. Blood 113:4637–4645

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Nielsen PS, Riber-Hansen R, Jensen TO, Schmidt H, Steiniche T (2013) Proliferation indices of phosphohistone H3 and Ki67: strong prognostic markers in a consecutive cohort with stage I/II melanoma. Mod Pathol 26(3):404–413

    Article  CAS  PubMed  Google Scholar 

  5. Colman H, Giannini C, Huang L, Gonzalez J, Hess K, Bruner J, Fuller G, Langford L, Pelloski C, Aaron J et al (2006) Assessment and prognostic significance of mitotic index using the mitosis marker phospho-histone H3 in low and intermediate-grade infiltrating astrocytomas. Am J Surg Pathol 30(5):657–664

    Article  PubMed  Google Scholar 

  6. Kim YJ, Ketter R, Steudel WI, Feiden W (2007) Prognostic significance of the mitotic index using the mitosis marker anti-phosphohistone H3 in meningiomas. Am J Clin Pathol 128(1):118–125

    Article  CAS  PubMed  Google Scholar 

  7. Nakashima S, Shiozaki A, Ichikawa D, Komatsu S, Konishi H, Iitaka D, Kubota T, Fujiwara H, Okamoto K, Kishimoto M et al (2013) Anti-phosphohistone H3 as an independent prognostic factor in human esophageal squamous cell carcinoma. Anticancer Res 33(2):461–467

    PubMed  Google Scholar 

  8. Ribalta T, McCutcheon IE, Aldape KD, Bruner JM, Fuller GN (2004) The mitosis-specific antibody anti-phosphohistone-H3 (PHH3) facilitates rapid reliable grading of meningiomas according to WHO 2000 criteria. Am J Surg Pathol 28(11):1532–1536

    Article  PubMed  Google Scholar 

  9. Fukushima S, Terasaki M, Sakata K, Miyagi N, Kato S, Sugita Y, Shigemori M (2009) Sensitivity and usefulness of anti-phosphohistone-H3 antibody immunostaining for counting mitotic figures in meningioma cases. Brain Tumor Pathol 26(2):51–57

    Article  PubMed  Google Scholar 

  10. Yafune A, Taniai E, Morita R, Akane H, Kimura M, Mitsumori K, Shibutani M (2014) Immunohistochemical cellular distribution of proteins related to M phase regulation in early proliferative lesions induced by tumor promotion in rat two-stage carcinogenesis models. Exp Toxicol Pathol 66(1):1–11

    Article  CAS  PubMed  Google Scholar 

  11. Yafune A, Taniai E, Morita R, Hayashi H, Suzuki K, Mitsumori K, Shibutani M (2013) Aberrant activation of M phase proteins by cell proliferation-evoking carcinogens after 28-day administration in rats. Toxicol Lett 219(3):203–210

    Article  CAS  PubMed  Google Scholar 

  12. Idriss MH, Kazlouskaya V, Malhotra S, Andres C, Elston DM (2013) Phosphohistone-H3 and Ki-67 immunostaining in cutaneous pilar leiomyoma and leiomyosarcoma (atypical intradermal smooth muscle neoplasm). J Cutan Pathol 40(6):557–563

    Article  PubMed  Google Scholar 

  13. Ladstein RG, Bachmann IM, Straume O, Akslen LA (2012) Prognostic importance of the mitotic marker phosphohistone H3 in cutaneous nodular melanoma. J Invest Dermatol 132(4):1247–1252

    Article  CAS  PubMed  Google Scholar 

  14. Wood MA, Hawk JD, Abel T (2006) Combinatorial chromatin modifications and memory storage: a code for memory? Learn Mem 13(3):241–244

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Wood MA, Kaplan MP, Brensinger CM, Guo W, Abel T (2005) Ubiquitin C-terminal hydrolase L3 (Uchl3) is involved in working memory. Hippocampus 15(5):610–621

    Article  CAS  PubMed  Google Scholar 

  16. Alarcon JM, Malleret G, Touzani K, Vronskaya S, Ishii S, Kandel ER, Barco A (2004) Chromatin acetylation, memory, and LTP are impaired in CBP+/- mice: a model for the cognitive deficit in Rubinstein-Taybi syndrome and its amelioration. Neuron 42(6):947–959

    Article  CAS  PubMed  Google Scholar 

  17. Korzus E, Rosenfeld MG, Mayford M (2004) CBP histone acetyltransferase activity is a critical component of memory consolidation. Neuron 42(6):961–972

    Article  CAS  PubMed  Google Scholar 

  18. Li J, Guo Y, Schroeder FA, Youngs RM, Schmidt TW, Ferris C, Konradi C, Akbarian S (2004) Dopamine D2-like antagonists induce chromatin remodeling in striatal neurons through cyclic AMP-protein kinase A and NMDA receptor signaling. J Neurochem 90(5):1117–1131

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  19. Crosio C, Heitz E, Allis CD, Borrelli E, Sassone-Corsi P (2003) Chromatin remodeling and neuronal response: multiple signaling pathways induce specific histone H3 modifications and early gene expression in hippocampal neurons. J Cell Sci 116(Pt 24):4905–4914

    Article  CAS  PubMed  Google Scholar 

  20. Hans F, Dimitrov S (2001) Histone H3 phosphorylation and cell division. Oncogene 20(24):3021–3027

    Article  CAS  PubMed  Google Scholar 

  21. Peterson CL, Laniel MA (2004) Histones and histone modifications. Curr Biol 14(14):R546–R551

    Article  CAS  PubMed  Google Scholar 

  22. Johansen KM, Johansen J (2006) Regulation of chromatin structure by histone H3S10 phosphorylation. Chromosome Res 14(4):393–404

    Article  CAS  PubMed  Google Scholar 

  23. Koshland D, Strunnikov A (1996) Mitotic chromosome condensation. Annu Rev Cell Dev Biol 12:305–333

    Article  CAS  PubMed  Google Scholar 

  24. Bradbury EM (1992) Reversible histone modifications and the chromosome cell cycle. Bioessays 14(1):9–16

    Article  CAS  PubMed  Google Scholar 

  25. Wei Y, Mizzen CA, Cook RG, Gorovsky MA, Allis CD (1998) Phosphorylation of histone H3 at serine 10 is correlated with chromosome condensation during mitosis and meiosis in Tetrahymena. Proc Natl Acad Sci U S A 95(13):7480–7484

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  26. Wei Y, Yu L, Bowen J, Gorovsky MA, Allis CD (1999) Phosphorylation of histone H3 is required for proper chromosome condensation and segregation. Cell 97(1):99–109

    Article  CAS  PubMed  Google Scholar 

  27. Gurley LR, D’Anna JA, Barham SS, Deaven LL, Tobey RA (1978) Histone phosphorylation and chromatin structure during mitosis in Chinese hamster cells. Eur J Biochem 84(1):1–15

    Article  CAS  PubMed  Google Scholar 

  28. Thomson S, Mahadevan LC, Clayton AL (1999) MAP kinase-mediated signalling to nucleosomes and immediate-early gene induction. Semin Cell Dev Biol 10(2):205–214

    Article  CAS  PubMed  Google Scholar 

  29. Van Hooser A, Goodrich DW, Allis CD, Brinkley BR, Mancini MA (1998) Histone H3 phosphorylation is required for the initiation, but not maintenance, of mammalian chromosome condensation. J Cell Sci 111(Pt 23):3497–3506

    PubMed  Google Scholar 

  30. Hendzel MJ, Wei Y, Mancini MA, Van Hooser A, Ranalli T, Brinkley BR, Bazett-Jones DP, Allis CD (1997) Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106(6):348–360

    Article  CAS  PubMed  Google Scholar 

  31. Prigent C, Dimitrov S (2003) Phosphorylation of serine 10 in histone H3, what for? J Cell Sci 116(Pt 18):3677–3685

    Article  CAS  PubMed  Google Scholar 

  32. Allis CD, Gorovsky MA (1981) Histone phosphorylation in macro- and micronuclei of Tetrahymena thermophila. Biochemistry 20(13):3828–3833

    Article  CAS  PubMed  Google Scholar 

  33. Hendzel MJ, Bazett-Jones DP (1997) Fixation-dependent organization of core histones following DNA fluorescent in situ hybridization. Chromosoma 106(2):114–123

    Article  CAS  PubMed  Google Scholar 

  34. Crosio C, Fimia GM, Loury R, Kimura M, Okano Y, Zhou H, Sen S, Allis CD, Sassone-Corsi P (2002) Mitotic phosphorylation of histone H3: spatio-temporal regulation by mammalian Aurora kinases. Mol Cell Biol 22(3):874–885

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  35. Angi M, Damato B, Kalirai H, Dodson A, Taktak A, Coupland SE (2011) Immunohistochemical assessment of mitotic count in uveal melanoma. Acta Ophthalmol 89(2):e155–e160

    Article  PubMed  Google Scholar 

  36. Fischer AH, Jacobson KA, Rose J, Zeller R (2008) Hematoxylin and eosin staining of tissue and cell sections. CSH Protoc 2008:pdb.prot4986

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Molecular Medicine, USF Health Byrd Alzheimer’s Institute, University of South Florida, 4001 E Fletcher Ave, Tampa, 33613, FL, USA

    Jaya Padmanabhan

Authors
  1. Jaya Padmanabhan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jaya Padmanabhan .

Editor information

Editors and Affiliations

  1. H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida, USA

    Srikumar P. Chellappan

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Padmanabhan, J. (2015). Immunostaining Analysis of Tissue Cultured Cells and Tissue Sections Using Phospho-Histone H3 (Serine 10) Antibody. In: Chellappan, S. (eds) Chromatin Protocols. Methods in Molecular Biology, vol 1288. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2474-5_13

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2474-5_13

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2473-8

  • Online ISBN: 978-1-4939-2474-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation