Enhancement of immunogold-labelled focal adhesion sites in fibroblasts cultured on metal substrates: problems and solutions.
Visualisation of cell adhesion patterns by scanning electron microscopy requires special preparation and labelling. The membranes and cytoplasm must be removed, without damaging the antigen, to facilitate antibody access to vinculin in the focal adhesions. Low beam energy imaging is used to visualise the cell undersurface (embedded in resin after staining with osmium tetroxide) and immunogold-labelled adhesion sites. The gold probe, must be large enough (>40 nm) for detection, while viewing the whole cell, but large gold markers increase steric hindrance and decrease labelling efficiency. This problem can be overcome by using small gold probes (1-5 nm) followed by enlargement with silver enhancement, but osmium tetroxide stain etches the silver. We demonstrated that metal substrates increased this etching. Reducing the concentration of osmium tetroxide and incubation time reduced the amount of etching. We have demonstrated that gold enhancement was not etched by osmium tetroxide, irrespective of the substrate. Therefore, comparative studies of cell adhesion to different biomaterial substrates can be performed using immunogold-labelling with gold enhancement.
Owen
G R
GR
AO Research Institute, Clavadelerstrasse, Davos Platz, Switzerland.
Meredith
D O
DO
Ap Gwynn
I
I
Richards
R G
RG
D000180:Acrylic Resins; D000818:Animals; D002448:Cell Adhesion; D002462:Cell Membrane / Q000648:ultrastructure*; D002478:Cells, Cultured; D005347:Fibroblasts / Q000502:physiology* / Q000648:ultrastructure; D022001:Focal Adhesions; D006046:Gold / Q000737:chemistry; D007150:Immunohistochemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D008855:Microscopy, Electron, Scanning / Q000379:methods; D016253:Microscopy, Immunoelectron / Q000379:methods; D009993:Osmium Tetroxide / Q000737:chemistry; D016613:Plastic Embedding; D012834:Silver / Q000737:chemistry; D013193:Stainless Steel / Q000737:chemistry; D016596:Vinculin / Q000378:metabolism
D000180:Acrylic Resins; D016596:Vinculin; D013193:Stainless Steel; D012834:Silver; D006046:Gold; C048707:LR white; D009993:Osmium Tetroxide