Environmental scanning electron microscopy gold immunolabeling in cell biology.

Francesco Rosso, Ferdinando Papale, Alfonso Barbarisi

IX Division of General Surgery and Applied Biotechnology, Department of Anasesthesological, Surgical and Emergency Sciences, Second University of Naples, Naples, Italy. francesco.rosso@unina2.it

Methods in molecular biology (Clifton, N.J.) 2013

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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.

Citation

Francesco Rosso, Ferdinando Papale, Alfonso Barbarisi. Environmental scanning electron microscopy gold immunolabeling in cell biology. Methods in molecular biology (Clifton, N.J.). 2013;931:517-23