Cell surface protein detection with immunogold labelling in ESEM: optimisation of the method and semi-quantitative analysis.
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.
Muscariello
Livio
L
IX Division of General Surgery and Applied Biotechnology, Department of Anaesthesological, Surgical and Emergency Sciences, Second University of Naples, Naples, Italy.
Rosso
Francesco
F
Marino
Gerardo
G
Barbarisi
Manlio
M
Cafiero
Gennaro
G
Barbarisi
Alfonso
A
D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D005347:Fibroblasts / Q000166:cytology / Q000648:ultrastructure; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D037502:Galectin 3 / Q000032:analysis* / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods*; D051379:Mice; D008855:Microscopy, Electron, Scanning
D037502:Galectin 3