Quantitative Analysis of In Vivo Cell Proliferation

Heather A. Cameron1

1 National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 3.9
DOI:  10.1002/N0471142301.ns0309s37
Online Posting Date:  November, 2006
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Injection and immunohistochemical detection of 5‐bromo‐2′‐deoxyuridine (BrdU) has become the standard method for studying the birth and survival of neurons, glia, and other cell types in the nervous system. BrdU, a thymidine analog, becomes stably incorporated into DNA during the S‐phase of mitosis. Because DNA containing BrdU can be specifically recognized by antibodies, this method allows dividing cells to be marked at any given time and then identified at time points from a few minutes to several years later. BrdU immunohistochemistry is suitable for cell counting to examine the regulation of cell proliferation and cell fate. It can be combined with labeling by other antibodies, allowing confocal analysis of cell phenotype or expression of other proteins. The potential for nonspecific labeling and toxicity are discussed. Although BrdU immunohistochemistry has almost completely replaced tritiated thymidine autoradiography for labeling dividing cells, this method and situations in which it is still useful are also described.

Keywords: proliferation; cell birth; cell division; neurogenesis; bromodeoxyuridine; thymidine; immunohistochemistry

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

Table of Contents

  • Basic Protocol 1: 5‐Bromo‐2′‐Deoxyuridine (BrdU) Immunohistochemistry
  • Alternate Protocol 1: 5‐Bromo‐2′‐Deoxyuridine (BrdU) Multiple‐Label Immunohistochemistry
  • Alternate Protocol 2: In Vivo [3H]Thymidine Autoradiography
  • Support Protocol 1: In Vivo Cell Labeling and Tissue Preparation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: 5‐Bromo‐2′‐Deoxyuridine (BrdU) Immunohistochemistry

  Materials
  • Tissue sections (see protocol 4)
  • 0.01 M citric acid in water, pH adjusted to 6.0 (prepare in advance and store at room temperature)
  • Trypsin buffer (see recipe)
  • PBS ( appendix 2A)
  • 2 N HCl in PBS (prepare in advance and store at room temperature)
  • Blocking solution (see recipe)
  • Mouse monoclonal anti‐BrdU antibody (BD Biosciences)
  • Biotinylated anti‐mouse secondary antibody (Sigma or Vector Labs)
  • 0.3% (v/v) hydrogen peroxide in PBS (store up to 1 year at 4°C)
  • Enzymatic detection kit (e.g., Elite Peroxidase ABC Kit, Vector Labs)
  • Peroxidase substrate for BrdU, preferably strong chromogenic substrate: e.g., cobalt‐enhanced diaminobenzidine (SigmaFast DAB tablets, Sigma) or VIP, Vector Labs
  • 0.1% (w/v) cresyl violet acetate in distilled water (store indefinitely at room temperature; filter before use)
  • 70% and 100% (v/v) ethanol
  • 0.25% (v/v) acetic acid in 95% ethanol (store indefinitely at room temperature)
  • Histological clearing agent (e.g., Americlear, Baxter; Histoclear, National Diagnostics; or Hemo‐De, Fisher)
  • Permanent histological mounting medium (e.g., Permount, Fisher)
  • Superfrost Plus slides (Fisher)
  • PAP pen
  • Microwave‐safe slide rack
  • Microwave‐safe staining dishes
  • Humidified chamber, e.g., a shallow 245‐mm2 polystyrene cell culture dish with wet paper towels on the bottom and double rows of 1‐ml pipets to hold slides above the paper towels
  • Coverslips

Alternate Protocol 1: 5‐Bromo‐2′‐Deoxyuridine (BrdU) Multiple‐Label Immunohistochemistry

  • Additional primary antibodies of interest
  • Fluorescently labeled secondary antibodies against mouse and other species used (Jackson Immunolabs or Molecular Probes)
  • Hoechst 33258 (bisbenzimide, 1 µg/ml in 10 mM sodium bicarbonate (store indefinitely at 4°C)
  • Aqueous hardening mounting medium (e.g., Immumount, Shandon or ProLong Gold, Molecular Probes)
  • 24‐well plates

Alternate Protocol 2: In Vivo [3H]Thymidine Autoradiography

  • Photographic emulsion suitable for detecting tritium (e.g., NTB, Eastman Kodak)
  • Photographic developer (e.g., Dektol, Eastman Kodak)
  • Fixer without ammonium hardener (e.g., Kodak Fixer, Eastman Kodak)
  • Gelatin‐subbed slides (unit 1.1)
  • Slide boxes
  • Darkroom with red or yellow safelight (i.e., 15‐W bulb with Eastman Kodak safelight filter no. 2 or Thomas Duplex Sodium Vapor Darkroom Safelight)
  • 40‐ to 80‐ml new glass beaker
  • 40° to 42°C water bath
  • Emulsion dipping vessel (e.g., Dip Miser, EM Science)
  • Plain (unsubbed) glass slides
  • Aluminum foil

Support Protocol 1: In Vivo Cell Labeling and Tissue Preparation

  Materials
  • Rat or mouse
  • Labeling reagent:
    • [3H]thymidine in sterile aqueous solution (e.g., methyl‐[3H]thymidine, 1 mCi/ml, 50 to 90 Ci/mmol, DuPont NEN)
    • 5‐bromo‐2′‐deoxyuridine (BrdU) labeling reagent (see recipe)
  • Anesthetic (e.g., isoflurane, Zeneca or pentobarbital; also see appendix 4B)
  • 4% (w/v) paraformaldehyde (see unit 1.1)
  • Sliding microtome or oscillating tissue slicer (e.g., OTS, EM Science; Vibratome, TPI; see unit 1.1)
  • Additional reagents and equipment for injection of rodents ( appendix 4F), anesthesia of rodents ( appendix 4B), perfusion fixation and tissue sectioning (unit 1.1)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Aten, J.A., Stap, J., Hoebe, R., and Bakker, P.J. 1994. Application and detection of IdUrd and CldUrd as two independent cell‐cycle markers. Methods Cell Biol. 41:317‐326.
   Bannigan, J. and Langman, J. 1979. The cellular effect of 5‐bromodeoxyuridine on the mammalian embryo. J. Embryol. Exp. Morphol. 50:123‐135.
   Baserga, R. and Malamud, D. 1969. Autoradiography: Techniques and Application. Harper and Row, New York.
   Biggers, W.J., Barnea, E.R., and Sanyal, M.K. 1987. Anomalous neural differentiation induced by 5‐bromo‐2′‐deoxyuridine during organogenesis in the rat. Teratology 35:63‐75.
   Burholt, D.R., Schultze, B., and Maurer, W. 1973. Autoradiographic confirmation of the mitotic division of every mouse jejunal crypt cell labeled with 3H‐thymidine. Evidence against the existence of cells synthesizing metabolic DNA. Cell Tissue Kinet. 6:229‐237.
   Cai, L., Hayes, N.L., and Nowakowski, R.S. 1997. Local homogeneity of cell cycle length in developing mouse cortex. J. Neurosci. 17:2079‐2087.
   Cameron, H.A. and McKay, R.D. 2001. Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus. J. Comp. Neurol. 435:406‐417.
   del Rio, J.A. and Soriano, E. 1989. Immunocytochemical detection of 5′‐bromodeoxyuridine incorporation in the central nervous system of the mouse. Brain Res. Dev. Brain Res. 49:311‐317.
   Drapeau, E., Mayo, W., Aurousseau, C., Le Moal, M., Piazza, P.V., and Abrous, D.N. 2003. Spatial memory performances of aged rats in the water maze predict levels of hippocampal neurogenesis. Proc. Natl. Acad. Sci. U.S.A. 100:14385‐14390.
   Ehmann, U.K., Williams, J.R., Nagle, W.A., Brown, J.A., Belli, J.A., and Lett, J.T. 1975. Perturbations in cell cycle progression from radioactive DNA precursors. Nature 258:633‐636.
   Feinendegen, L.E. 1967. Tritium‐Labeled Molecules in Biology and Medicine. Academic Press, New York.
   Kolb, B., Pedersen, B., Ballermann, M., Gibb, R., and Whishaw, I.Q. 1999. Embryonic and postnatal injections of bromodeoxyuridine produce age‐dependent morphological and behavioral abnormalities. J. Neurosci. 19:2337‐2346.
   Likhachev, A.J., Tomatis, L., and Margison, G.P. 1983. Incorporation and persistence of 5‐bromodeoxyuridine in newborn rat tissue DNA. Chem. Biol. Interact. 46:31‐38.
   Mares, V., Schultze, B., and Maurer, W. 1974. Stability of DNA in Purkinje cell nuclei of the mouse. J. Cell Biol. 63:665‐674.
   Maslov, A.Y., Barone, T.A., Plunkett, R.J., and Pruitt, S.C. 2004. Neural stem cell detection, characterization, and age‐related changes in the subventricular zone of mice. J. Neurosci. 24:1726‐1733.
   Miller, M.W. and Nowakowski, R.S. 1988. Use of bromodeoxyuridine‐immunohistochemistry to examine the proliferation, migration and time of origin of cells in the central nervous system. Brain Res. 457:44‐52.
   Packard, D.S. Jr., Menzies, R.A., and Skalko, R.G. 1973. Incorporation of thymidine and its analogue, bromodeoxyuridine, into embryos and maternal tissues of the mouse. Differentiation 1:397‐404.
   Palmer, T.D., Willhoite, A.R., and Gage, F.H. 2000. Vascular niche for adult hippocampal neurogenesis. J. Comp. Neurol. 425:479‐494.
   Parent, J.M., Tada, E., Fike, J.R., and Lowenstein, D.H. 1999. Inhibition of dentate granule cell neurogenesis with brain irradiation does not prevent seizure‐induced mossy fiber synaptic reorganization in the rat. J. Neurosci. 19:4508‐4519.
   Pelc, S.R. 1972. Metabolic DNA in ciliated protozoa, salivary gland chromosomes and mammalian cells. Int. Rev. Cytol. 32:327‐355.
   Post, J., Huang, C.Y., and Hoffman, J. 1963. The replication time and pattern of the liver cell in the growing rat. J. Cell Biol. 18:1‐12.
   Rogers, A.W. 1973. Techniques of Autoradiography. Elsevier/North‐Holland, Amsterdam.
   Sekerkova, G., Ilijic, E., and Mugnaini, E. 2004. Bromodeoxyuridine administered during neurogenesis of the projection neurons causes cerebellar defects in rat. J. Comp. Neurol. 470:221‐239.
   Takahashi, T., Nowakowski, R.S., and Caviness, V.S. Jr. 1992. BUdR as an S‐phase marker for quantitative studies of cytokinetic behaviour in the murine cerebral ventricular zone. J. Neurocytol. 21:185‐197.
   Wintzerish, M., Witterndorp, E., Rechenmann, R.V., and Mandel, P. 1977. Nuclear, nucleolar repair, or turnover of DNA in adult rat brain. J. Neurosci. Res. 3:217‐230.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library