Focal adhesion interactions with topographical structures: a novel method for immuno-SEM labelling of focal adhesions in S-phase cells.
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.
Biggs
M J P
MJ
Centre for Cell Engineering, Institute of Biomedical and Life Sciences, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, United Kingdom. m.biggs.1@research.gla.ac.uk
Richards
R G
RG
Wilkinson
C D W
CD
Dalby
M J
MJ
D000369:Aged, 80 and over; D002448:Cell Adhesion; D002453:Cell Cycle; D002478:Cells, Cultured; D005260:Female; D005269:Femur / Q000166:cytology*; D022001:Focal Adhesions; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D010006:Osteoblasts / Q000166:cytology / Q000648:ultrastructure*; D016196:S Phase / Q000276:immunology / Q000502:physiology