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

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.