Detection of Cell Proliferation Markers by Immunofluorescence Staining and Microscopy Imaging in Paraffin‐Embedded Tissue Sections

Seda Eminaga1, Polakit Teekakirikul2, Christine E. Seidman3, Jonathan G. Seidman3

1 Cardiovascular Division, King's College London, London, 2 Heart and Vascular Institute, Pittsburgh, UPMC Presbyterian, 3 Department of Genetics, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 14.25
DOI:  10.1002/cpmb.13
Online Posting Date:  July, 2016
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit describes a step‐by‐step protocol to detect and quantify proliferating cells in paraffin‐embedded tissue sections. Two well‐established markers of proliferation (incorporation of BrdU into newly synthesized DNA and expression of the nuclear protein Ki67) are detected after antigen‐retrieval and subsequent immunofluorescence staining and confocal microscopy. © 2016 by John Wiley & Sons, Inc.

Keywords: BrdU; cell proliferation; immunofluorescence; immunohistochemistry; Ki67

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Immunofluorescent Detection of BrdU or Ki67 in Paraffin‐Embedded Tissue Sections
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Immunofluorescent Detection of BrdU or Ki67 in Paraffin‐Embedded Tissue Sections

  Materials
  • 5‐μm thick paraffin‐embedded tissue sections mounted on positively charged glass slides (Superfrost Plus, Thermo Scientific, cat. no. J1800AMNZ)
  • Xylene (histological grade, Sigma)
  • 70%, 80%, 95%, and 100% (v/v) ethanol (histological grade, Sigma)
  • Phosphate‐buffered saline (PBS; appendix 22)
  • Tween‐20 (Sigma, cat. no. P9416)
  • Antigen retrieval buffer (see recipe)
  • Tris‐buffered saline (TBS, see recipe)
  • 100,000 U/ml exonuclease III (Exo III, New England Biolabs, cat. no. M0206S)
  • 20,000 U/ml EcoRI and 10× EcoRI buffer (New England Biolabs, cat. no. R0101S)
  • TBS with 0.1% (v/v) Tween‐20 (TBS‐T)
  • Blocking buffer: TBS containing 10% normal goat serum (Vector Laboratories, cat. no. S‐1000)
  • Antibody diluent (Dako, cat. no. S0809)
  • Primary monoclonal antibody:
  • Mouse anti‐BrdU clone Bu20a (Dako, cat. no. M0744)
  • Rat anti‐BrdU clone BU1/75 (ICR1) (Abcam, cat. no. ab6326)
  • Rat anti‐Ki67 clone SolA15 (eBioscience, cat. no. 14‐5698‐82)
  • Species‐matched IgG: mouse IgG1 (Dako, cat. no. X0931) or rat IgG (Vector Laboratories, cat. no. I‐4000)
  • Alexa Fluor 488–conjugated goat anti‐mouse or anti‐rat secondary antibody (Life Technologies, cat. nos. A11001 or A11006)
  • Mounting reagent containing DAPI (Prolong Gold Antifade Mountant with DAPI, Life Technologies, cat. no. P36935)
  • Staining racks and vessels with lids (xylene‐ and heat‐resistant; Tissue‐Tek Slide Staining Set, Sakura Finetek)
  • Programmable pressure‐cooker (e.g., Biocare Medical Decloaking Chamber) or alternative heating device (e.g., water bath, microwave oven, vegetable steamer)
  • Rocker that can accommodate a staining dish
  • Low‐lint wipes (e.g., VWR, cat. no. 115‐0202)
  • Hydrophobic pen (Dako, cat. no. S2002)
  • Humidified chamber: commercial (e.g., StainTray, Simport, cat. no. M920) or prepared as in Figure
  • 37° and 60°C incubators
  • Aluminum foil
  • Glass coverslips (VWR, cat. no. 631‐9430)
  • Fluorescent confocal microscope with standard FITC filter (peak excitation, 490 nm; peak emission, 526 nm)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Brown, J.K., Pemberton, A.D., Wright, S.H., and Miller, H.R. 2004. Primary antibody‐Fab fragment complexes: A flexible alternative to traditional direct and indirect immunolabeling techniques. J. Histochem. Cytochem. 52:1219‐1230. doi: 10.1369/jhc.3A6200.2004.
  Dinjens, W.N., ten Kate, J., Lenders, M.H., van der Linden, E.P., and Bosman, F.T. 1992. Bromodeoxyuridine (BrdU) immunocytochemistry by exonuclease III (Exo III) digestion. Histochemistry 98:199‐205. doi: 10.1007/BF00315878.
  Gomez‐Nicola, D., Valle‐Argos, B., Pallas‐Bazarra, N., and Nieto‐Sampedro, M. 2011. Interleukin‐15 regulates proliferation and self‐renewal of adult neural stem cells. Mol. Biol. Cell 22:1960‐1970. doi: 10.1091/mbc.E11‐01‐0053.
  Goodson, W.H. 3rd, Moore, D.H. 2nd, Ljung, B.M., Chew, K., Florendo, C., Mayall, B., Smith, H.S., and Waldman, F.M. 1998. The functional relationship between in vivo bromodeoxyuridine labeling index and Ki‐67 proliferation index in human breast cancer. Breast Cancer Res. Treat. 49:155‐164. doi: 10.1023/A:1005926228093.
  Goto, H., Tomono, Y., Ajiro, K., Kosako, H., Fujita, M., Sakurai, M., Okawa, K., Iwamatsu, A., Okigaki, T., Takahashi, T., and Inagaki, M. 1999. Identification of a novel phosphorylation site on histone H3 coupled with mitotic chromosome condensation. J. Biol. Chem. 274:25543‐25549. doi: 10.1074/jbc.274.36.25543.
  Gratzner, H.G. 1982. Monoclonal antibody to 5‐bromo‐ and 5‐iododeoxyuridine: A new reagent for detection of DNA replication. Science 218:474‐475. doi: 10.1126/science.7123245.
  Hendzel, M.J., Wei, Y., Mancini, M.A., Van Hooser, A., Ranalli, T., Brinkley, B.R., Bazett‐Jones, D.P., and Allis, C.D. 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 106:348‐360. doi: 10.1007/s004120050256.
  Hughes, W.L., Bond, V.P., Brecher, G., Cronkite, E.P., Painter, R.B., Quastler, H., and Sherman, F.G. 1958. Cellular proliferation in the mouse as revealed by autoradiography with tritiated thymidine. Proc. Natl. Acad. Sci. U.S.A. 44:476‐483. doi: 10.1073/pnas.44.5.476.
  Ivell, R., Teerds, K., and Hoffman, G.E. 2014. Proper application of antibodies for immunohistochemical detection: Antibody crimes and how to prevent them. Endocrinology 155:676‐687. doi: 10.1210/en.2013‐1971.
  Jecker P., Beuleke, A., Dressendorfer, I., Pabst, R., and Westermann, J. 1997. Long‐term oral application of 5‐bromo‐2‐deoxyuridine does not reliably label proliferating immune cells the the LEW rat. J. Histochem. Cytochem. 45:393‐401. doi: 10.1177/002215549704500307.
  Kao, G.D., McKenna, W.G., and Yen, T.J. 2001. Detection of repair activity during the DNA damage‐induced G2 delay in human cancer cells. Oncogene 20:3486‐3496. doi: 10.1038/sj.onc.1204445.
  Kee, N., Sivalingam, S., Boonstra, R., and Wojtowicz, J.M. 2002. The utility of Ki‐67 and BrdU as proliferative markers of adult neurogenesis. J. Neurosci. Methods 115:97‐105. doi: 10.1016/S0165‐0270(02)00007‐9.
  Limsirichaikul, S., Niimi, A., Fawcett, H., Lehmann, A., Yamashita, S., and Ogi, T. 2009. A rapid non‐radioactive technique for measurement of repair synthesis in primary human fibroblasts by incorporation of ethynyl deoxyuridine (EdU). Nucleic Acids Res. 37:e31. doi: 10.1093/nar/gkp023.
  Montuenga, L.M., Springall, D.R., Gaer, J., McBride, J.T., and Polak, J.M. 1992. Simultaneous immunostaining method for localization of bromodeoxyuridine and calcitonin gene‐related peptide. J. Histochem. Cytochem. 40:1121‐1128. doi: 10.1177/40.8.1377732.
  Muskhelishvili, L., Latendresse, J.R., Kodell, R.L., and Henderson, E.B. 2003. Evaluation of cell proliferation in rat tissues with BrdU, PCNA, Ki‐67(MIB‐5) immunohistochemistry and in situ hybridization for histone mRNA. J. Histochem. Cytochem. 51:1681‐1688. doi: 10.1177/002215540305101212.
  Salic, A. and Mitchison, T.J. 2008. A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc. Natl. Acad. Sci. U.S.A. 105:2415‐2420. doi: 10.1073/pnas.0712168105.
  Sarli, G., Benazzi, C., Preziosi, R., and Marcato, P.S. 1995. Assessment of proliferative activity by anti‐PCNA monoclonal antibodies in formalin‐fixed, paraffin‐embedded samples and correlation with mitotic index. Vet. Pathol. 32:93‐96. doi: 10.1177/030098589503200120.
  Scholzen, T. and Gerdes, J. 2000. The Ki‐67 protein: From the known and the unknown. J. Cell. Physiol. 182:311‐322. doi: 10.1002/(SICI)1097‐4652(200003)182:3%3c311::AID‐JCP1%3e3.0.CO;2‐9.
  Scott, R.J., Hall, P.A., Haldane, J.S., van Noorden, S., Price, Y., Lane, D.P., and Wright, N.A. 1991. A comparison of immunohistochemical markers of cell proliferation with experimentally determined growth fraction. J. Pathol. 165:173‐178. doi: 10.1002/path.1711650213.
  Shi, S.R., Shi, Y., and Taylor, C.R. 2011. Antigen retrieval immunohistochemistry: Review and future prospects in research and diagnosis over two decades. J. Histochem. Cytochem. 59:13‐32. doi: 10.1369/jhc.2010.957191.
  Teekakirikul, P., Eminaga, S., Toka, O., Alcalai, R., Wang, L., Wakimoto, H., Nayor, M., Konno, T., Gorham, J.M., Wolf, C.M., Kim, J.B., Schmitt, J.P., Molkentin, J.D., Norris, R.A., Tager, A.M., Hoffman, S.R., Markwald, R.R., Seidman, C.E., and Seidman, J.G. 2010. Cardiac fibrosis in mice with hypertrophic cardiomyopathy is mediated by non‐myocyte proliferation and requires Tgf‐beta. J. Clin. Invest. 120:3520‐3529. doi: 10.1172/JCI42028.
  Van Hooser, A., Goodrich, D.W., Allis, C.D., Brinkley, B.R., and Mancini, M.A. 1998. Histone H3 phosphorylation is required for the initiation, but not maintenance, of mammalian chromosome condensation. J. Cell Sci. 111:3497‐3506.
  Zeller, R. 2001. Fixation, embedding, and sectioning of tissue, embryos, and single cells. Curr. Protoc. Mol. Biol. 7:14.1.1‐14.1.8.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library