Optimal processing method to obtain four-color confocal fluorescent images of the cytoskeleton and nucleus in three-dimensional chondrocyte cultures

被引:10
作者
Blanc, A
Tran-Khanh, N
Filion, D
Buschmann, MD
机构
[1] Ecole Polytech, Dept Chem Engn, Montreal, PQ H3C 3A7, Canada
[2] Ecole Polytech, Inst Biomed Engn, Montreal, PQ H3C 3A7, Canada
关键词
cartilage; chondrocyte; cytoskeleton; confocal microscopy; actin; tubulin; vimentin;
D O I
10.1369/jhc.5B6728.2005
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
Tissue engineering of articular cartilage requires accurate imaging of the chondrocyte cytoskeleton. Past studies have applied various fixation and permeabilization protocols without optimization of parameters. In this study, we have examined procedures using glutaralclehyde and paraformaldehyde as fixatives and Triton X-100 and Octyl-POE as permeabilizing detergents. A four-color fluorescence confocal method was developed to simultaneously image actin, tubulin, vimentin, and the nucleus. We found optimal preservation and morphology of the chondrocyte cytoskeleton after simultaneous fixation and permeabilization with glutaralclehyde and Triton X-100. These images displayed less cellular shrinkage and higher-resolution filamentous structures than with paraformaldehyde or when permeabilization followed fixation.
引用
收藏
页码:1171 / 1175
页数:5
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