Advertisement

Environmental Scanning Electron Microscopy Gold Immunolabeling in Cell Biology

  • Francesco Rosso
  • Ferdinando Papale
  • Alfonso Barbarisi
Part of the Methods in Molecular Biology book series (MIMB, volume 931)

Abstract

Immunogold labeling (IGL) technique has been utilized by many authors in combination with scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to obtain the identification/localization of receptors and antigens, both in cells and tissues. Environmental scanning electron microscopy (ESEM) represents an important tool in biomedical research, since it does not require any severe processing of the sample, lowering the risk of generating artifacts and interfere with the IGL procedure. The absence of metal coating could yield further advantages for our purpose as the labeling detection is based on the atomic number difference between nanogold spheres and the biological material. Using the gaseous secondary electron detector, compositional contrast is easily revealed by the backscattered electron component of the signal. In spite of this fact, only few published papers present a combination of ESEM and IGL. Hereby we present our method, optimized to improve the intensity and the specificity of the labeling signal, in order to obtain a semiquantitative evaluation of the labeling signal.

In particular, we used a combination of IGL and ESEM to detect the presence of a protein on the cell surface. To achieve this purpose, we chose as an experimental system 3T3 Swiss albino mouse fibroblasts and galectin-3.

Key words

Immunogold labeling Environmental scanning electron microscopy Surface protein detection Silver enhancement reaction 

Notes

Acknowledgement

This research has been funded through the Ministry of Instruction, University and Research (MIUR), number 200734RMKE: Validation of molecular markers and technology innovation in environmental scanning electron microscopy (ESEM) for the clinical and prognostic setting of clonal proliferative thyroid lesions.

References

  1. 1.
    Faulk W, Taylor G (1979) An immunocolloid method for the electron microscope. Immunochemistry 8:1081–1083CrossRefGoogle Scholar
  2. 2.
    Hoyer LC, Lee JC, Bucana C (1979) Scanning immunoelectron microscopy for the identification and mapping of two or more antigens on cell surfaces. Scan Electron Microsc 3:629–636PubMedGoogle Scholar
  3. 3.
    De Harven E, Leung R, Christensen H (1984) A novel approach for scanning electron microscopy of colloidal gold labelled cell surfaces. J Cell Biol 99:53–57PubMedCrossRefGoogle Scholar
  4. 4.
    Williams TE, Nagarajan S, Selvaraj P, Zhu C (2001) Quantifying the impact of membrane microtopology on effective two-dimensional affinity. J Biol Chem 276:13283–13288PubMedCrossRefGoogle Scholar
  5. 5.
    Bendayan M (1995) Colloidal gold post-embedding immunocytochemistry. Prog Histochem Cytochem 2:1–159Google Scholar
  6. 6.
    Polok JM, Van Noorden S (1986) Immunocytochemistry: modern methods and applications, 2nd edn. John Wright, BristolGoogle Scholar
  7. 7.
    Martinez-Alvarez C, Tudela C, Perez-Miguelsanz J, O’Kane S, Puerta J, Ferguson MW (2000) Medial edge epithelial cell fate during palatal fusion. Dev Biol 220:343–357PubMedCrossRefGoogle Scholar
  8. 8.
    Mestres P, PutzN LaueM (2003) Applications of ESEM to the study of biomedical specimens. Microsc Microanal 9:490–491Google Scholar
  9. 9.
    Muscariello L, Rosso F, Marino G, Giordano A, Barbarisi M, Cafiero G, Barbarisi A (2005) A critical overview of ESEM biological applications. J Cell Physiol 205:328–334PubMedCrossRefGoogle Scholar
  10. 10.
    Rosso F, Marino G, Muscariello L, Cafiero G, Favia P, D’Aloia E, D’Agostino R, Barbarisi A (2006) Adhesion and proliferation of fibroblasts on plasma-deposited nanostructured fluorocarbon coatings: evidence of FAK activation. J Cell Physiol 207:636–643PubMedCrossRefGoogle Scholar
  11. 11.
    McMenamin PG, Djano J, Wealthall R, Griffin BJ (2002) Characterization of the macrophages associated with the tunica vasculosa lentis of the rat eye. Invest Ophthalmol Vis Sci 43:2076–2082PubMedGoogle Scholar
  12. 12.
    Ramandeep DKL, Raje M (2001) Optimization of immunogold labeling TEM: an ELISA based method for rapid and convenient simulation of processing conditions for quantitative detection of antigen. J Histochem Cytochem 49:355–367PubMedCrossRefGoogle Scholar
  13. 13.
    Soltys BJ, Andrews DW, Jemmerson R, Gupta RS (2001) Cytochrome-c localizes in secretory granules in pancreas and anterior pituitary. Cell Biol Int 25:331–338PubMedCrossRefGoogle Scholar
  14. 14.
    Tomczok J, Sliwa-Tomczok W, Klein CL, Bittinger F, Kirkpatrick CJ (1994) Application of immunogold labelling for light and electron microscopic localization of endothelial leukocyte adhesion molecule 1 (ELAM-1) on cultured human endothelial cells. Micron 25:257–266PubMedCrossRefGoogle Scholar
  15. 15.
    Muscariello L, Rosso F, Marino G, Barbarisi M, Cafiero G, Barbarisi A (2008) Cell surface protein detection with immunogold labelling in ESEM: optimisation of the method and semi-quantitative analysis. J Cell Physiol 214(3):769–776PubMedCrossRefGoogle Scholar
  16. 16.
    Krezslak A, Lipinska A (2004) Galectin-3 as a multifunctional protein. Cell Mol Biol Lett 9:305–328Google Scholar
  17. 17.
    Hayat MA (1989) Colloidal gold: principles, methods and applications, vols 1 & 2. Academic, San DiegoGoogle Scholar
  18. 18.
    Namork E, Heier HE (1989) Silver enhancement of gold probes (5–40 nm): single and double labeling of antigenic sites on the surfaces imaged with backscattered electrons. J Electron Microsc Tech 11:102–108PubMedCrossRefGoogle Scholar
  19. 19.
    Cafiero G, Papale F, Grimaldi A, Rosso F, Barbarisi M, Tortora C, Marino G, Barbarisi A (2011) Immunogold labelling in environmental scanning electron microscopy: applicative features for complementary cytological interpretation. J Microsc 241(1):83–93PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Francesco Rosso
    • 1
  • Ferdinando Papale
    • 1
  • Alfonso Barbarisi
    • 1
  1. 1.IX Division of General Surgery and Applied Biotechnology, Department of Anasesthesological, Surgical and Emergency SciencesSecond University of NaplesNaplesItaly

Personalised recommendations