THE ULTRASTRUCTURE OF ANIONIC SITES IN RAT ARTICULAR-CARTILAGE AS REVEALED BY DIFFERENT PREPARATION METHODS AND POLYETHYLENEIMINE STAINING

The ultrastructure of anionic sites in the middle layer of rat articul ar cartilages was studied by two methods, the quick-freezing and deep- etching method, and the quick-freezing and freeze-substitution method. The anionic sites were visualized with a cationic tracer, polyethylen eimine. They were also compared with those revealed in tissues subject ed to conventional fixation, such as pre-embedding or post-embedding. With the deep-etching method, three-dimensional meshwork structures we re observed more clearly in the extracellular matrix compared with tho se seen in conventional ultrathin sections. In combination with polyet hyleneimine staining, in which no chemical contrast was needed for vis ualization of anionic sites, numerous stained particles were detected around filaments in the extracellular matrix, indicating that they wer e anionic sites consisting mainly of proteoglycans. With the pre-embed ding method and polyethyleneimine staining, the shapes of aggregated s tained particles varied with different preparation procedures, includi ng chemical fixation and contrasting. The fine meshworks were also obs erved with the post-embedding method and polyethyleneimine staining. I t is suggested that such images of anionic sites, as revealed by the d eep-etching method and the post-embedding polyethyleneimine-staining m ethod with low-temperature dehydration, are probably closer to native states than those revealed by the conventional pre-embedding polyethyl eneimine-staining method. (C) 1998 Chapman & Hall.