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Cell surface protein detection with immunogold labelling in ESEM: Optimisation of the method and semi‐quantitative analysis
Author(s) -
Muscariello Livio,
Rosso Francesco,
Marino Gerardo,
Barbarisi Manlio,
Cafiero Gennaro,
Barbarisi Alfonso
Publication year - 2008
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.21270
Subject(s) - immunogold labelling , environmental scanning electron microscope , labelling , biophysics , biological system , cell , chemistry , nanotechnology , biomedical engineering , microbiology and biotechnology , electron microscope , scanning electron microscope , materials science , biology , biochemistry , optics , physics , medicine , composite material
In this work we used a combination of immunogold labelling (IGL) and environmental scanning electron microscopy (ESEM) to detect the presence of a protein on the cell surface. To achieve this purpose we chose as experimental system 3T3 Swiss Albino Mouse Fibroblasts and galectin‐3. This protein, whose sub‐cellular distribution is still under discussion, is involved in a large number of cell physiological and pathological processes. IGL technique has been utilised by many authors in combination with SEM and TEM to obtain the identification/localisation of receptors and antigens, both in cells and tissues. ESEM represents an important tool in biomedical research, since it does not require any severe processing of the sample, lowering the risk of generating artefacts and interfere with IGL procedure. The absence of metal coating could yield further advantages for our purpose as the labelling detection is based on the atomic number difference between Nanogold spheres and the biological material. Using the gaseous secondary electron detector (GSED) compositional contrast is easily revealed by the backscattered electrons 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, optimised to improve the intensity and the specificity of the labelling signal, in order to obtain a semi‐quantitative evaluation of the labelling signal. J. Cell. Physiol. 214: 769–776, 2008. © 2007 Wiley‐Liss, Inc.