Ultrathin cryosections in the plane of cell monolayers: evaluation of their potential for antibody localization studies of the cytoskeleton.
In spite of the fact that the preembedding method is satisfactory for the ultrastructural localization of cytoskeletal proteins, there is a need for a localization method that retains the cells' ground substance, delicate filament arrangements, and membrane-filament interactions and provides a good delineation of ultrastructural detail. Ultracryomicrotomy, a resinless sectioning method, can combine good morphology with optimal antibody labeling. Until now, however, it has not been possible to section cell monolayers parallel to their plane of growth. This is a prerequisite for the localization of proteins along segments of filaments, contained within the section thickness. We describe such a method and give a first appreciation of its potential for antibody localization studies of cytoskeletal proteins. The method consists of seeding cells on a parallel 0.75-mm-thick gelatin substrate that can later be cut and used as a mounting block. An adapted negative staining has yielded a very useful delineation of the well-preserved structures within the cells, even in combination with immunogold labeling. The latter has been in its indirect version less satisfactory in dense microfilament bundles because of penetration problems, and more satisfactory on microtubules. Clearly, the penetration properties of gold probes will have to be improved before this method will become widely applicable. The availability of a sectioning method like this will provide the basis for further progress. There will be many cases which will justify the use of this relatively more difficult approach.
Langanger
G
G
Department of Life Sciences, Janssen Pharmaceutica, Beerse, Belgium.
De Mey
J
J
D002021:Buffers; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000032:analysis* / Q000648:ultrastructure; D005404:Fixatives; D005629:Frozen Sections; D005780:Gelatin; D007150:Immunohistochemistry; D013194:Staining and Labeling; D013395:Sucrose
D002021:Buffers; D005404:Fixatives; D013395:Sucrose; D005780:Gelatin