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 glutaraldehyde 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 glutaraldehyde and Triton X-100. These images displayed less cellular shrinkage and higher-resolution filamentous structures than with paraformaldehyde or when permeabilization followed fixation.
Blanc
Antoine
A
Department of Chemical Engineering, Ecole Polytechnique, PO 6079, Station Centre-ville, Montreal, QC, Canada H3C 3A7.
Tran-Khanh
Nicolas
N
Filion
Dominic
D
Buschmann
Michael D
MD
D000199:Actins / Q000378:metabolism; D000818:Animals; D002358:Cartilage, Articular / Q000378:metabolism / Q000648:ultrastructure; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism / Q000648:ultrastructure*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism / Q000648:ultrastructure*; D003599:Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure*; D005404:Fixatives; D005455:Fluorescent Antibody Technique; D005557:Formaldehyde; D005976:Glutaral; D020100:Hydrogels; D018613:Microscopy, Confocal; D017830:Octoxynol; D011108:Polymers; D012685:Sepharose; D016707:Tissue Fixation; D014404:Tubulin / Q000378:metabolism; D014746:Vimentin / Q000378:metabolism
D000199:Actins; D005404:Fixatives; D020100:Hydrogels; D011108:Polymers; D014404:Tubulin; D014746:Vimentin; D005557:Formaldehyde; D017830:Octoxynol; D012685:Sepharose; D005976:Glutaral; C003043:paraform