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JHC exPRESS: First Published June 2, 2005. doi:10.1369/jhc.5B6728.2005
Copyright © Histochemical Society, Inc.

A more recent version of this article appeared on September 1, 2005.
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Brief Reports

Optimal Processing Method to Obtain Four-color Confocal Fluorescent Images of the Cytoskeleton and Nucleus in Three-dimensional Chondrocyte Cultures

Antoine Blanc 1, Nicolas Tran-Khanh 1, Dominic Filion 1 and Michael D. Buschmann 1*

1 Institute of Biomedical Engineering (NT-K,DF,MDB) and Department of Chemical Engineering (AB,MDB), Ecole Polytechnique, Montreal, Quebec, Canada

* To whom correspondence should be addressed. E-mail: michael.buschmann{at}polymtl.ca.

Submitted on April 29, 2005
Accepted on 11 May 2005


  Abstract
Tissue engineering of articular cartilage requires accurate imaging of the chondrocyte cytoskeleton. Past studies have applied various fixation and permeabilisation protocols without optimization of parameters. Here we have examined procedures using glutaraldehyde and paraformaldehyde as fixatives and Triton-X-100 and Octyl-POE as permeabilising 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 glutaraldehyde and Triton X-100. These images displayed less cellular shrinkage and higher resolution filamentous structures than with paraformaldehyde or when permeabilisation followed fixation.

Key Words: cartilage, chondrocyte cytoskeleton, confocal, microscopy, actin, tubulin, vimentin


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