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)

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 > School of Molecular Biosciences
• Journal Name: Journal of Microscopy
• ISSN: 0022-2720