Focal adhesion interactions with topographical structures: a novel method for immuno-SEM labelling of focal adhesions in S-phase cells

Biggs, M.J., Richards, R.G., Wilkinson, C.D. and Dalby, M.J. (2008) Focal adhesion interactions with topographical structures: a novel method for immuno-SEM labelling of focal adhesions in S-phase cells. Journal of Microscopy, 231(1), pp. 28-37. (doi:10.1111/j.1365-2818.2008.02013.x)

Full text not currently available from Enlighten.

Publisher's URL: http://dx.doi.org/10.1111/j.1365-2818.2008.02013.x

Abstract

Current understanding of the mechanisms involved in osseointegration following implantation of a biomaterial has led to adhesion quantification being implemented as an assay of cytocompatibility. Such measurement can be hindered by intra-sample variation owing to morphological changes associated with the cell cycle. Here we report on a new scanning electron microscopical method for the simultaneous immunogold labelling of cellular focal adhesions and S-phase nuclei identified by BrdU incorporation. Prior to labelling, cellular membranes are removed by tritonization and antigens of non-interest blocked by serum incubation. Adhesion plaque-associated vinculin and S-phase nuclei were both separately labelled with a 1.4 nm gold colloid and visualized by subsequent colloid enhancement via silver deposition. This study is specifically concerned with the effects microgroove topographies have on adhesion formation in S-phase osteoblasts. By combining backscattered electron (BSE) imaging with secondary electron (SE) imaging it was possible to visualize S-phase nuclei and the immunogold-labelled adhesion sites in one energy 'plane' and the underlying nanotopography in another. Osteoblast adhesion to these nanotopographies was ascertained by quantification of adhesion complex formation.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Dalby, Professor Matthew
Authors: Biggs, M.J., Richards, R.G., Wilkinson, C.D., and Dalby, M.J.
Subjects:Q Science > QR Microbiology > QR180 Immunology
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Journal of Microscopy
ISSN:0022-2720

University Staff: Request a correction | Enlighten Editors: Update this record