Premium
IMMUNOGOLD LABELLING OF FIBROBLAST FOCAL ADHESION SITES VISUALISED IN FIXED MATERIAL USING SCANNING ELECTRON MICROSCOPY, AND LIVING, USING INTERNAL REFLECTION MICROSCOPY
Author(s) -
Richards R. G.,
Stiffanic M.,
Owen G. Rh,
Riehle M.,
Gwynn Ap I.,
Curtis A. S. G.
Publication year - 2001
Publication title -
cell biology international
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.932
H-Index - 77
eISSN - 1095-8355
pISSN - 1065-6995
DOI - 10.1006/cbir.2001.0807
Subject(s) - immunogold labelling , vinculin , microscopy , focal adhesion , substrate (aquarium) , materials science , electron microscope , scanning electron microscope , biophysics , chemistry , cell , pathology , optics , biology , biochemistry , medicine , ecology , physics , composite material
A new immunogold labelling method for the visualisation of vinculin, an integral protein in focal adhesions of cells, is reported. Quantification of vinculin is indicative of substrate cytocompatibility (cytocompatibility is one aspect of biocompatibility; it is the cellular response to a biomaterial). For efficient labelling, most of the cell body above the cell—substrate interface was removed with detergent. The antigen blocking procedure, size of label (5nm) and duration of silver‐enhancement (6min), for visualisation of the labelled sites on the whole cell by scanning electron microscopy (SEM), were determined. Imaging living cells with interference reflection light microscopy, followed by backscattered electron (BSE) imaging of the same fixed and immunolabelled cells confirmed the results. Collecting low voltage BSE images of embedded cells after the substrate had been removed provided ‘sectional’ views through the cell. This enabled visualisation of vinculin exclusively within the cell‐substrate contact zone; the focal adhesions. The method could be of general use in the imaging of protein distribution at biological tissue/substrate interfaces.