Preservation of cartilage matrix proteoglycans using cationic dyes chemically related to ruthenium hexaammine trichloride

Abstract

We tested various cationic dyes chemically related to ruthenium hexaammine trichloride (RHT) [i.e., the RHT-cyclohexanedione complex (RHT-CC), pentaamine ruthenium N-dimethylphenylenediimine trichloride (PRT), tris(bipyridyl)ruthenium(II) chloride (TRC), tris(bipyridyl)iron(II) chloride (TIC), and cobalt hexaammine trichloride (CHT)] for their effectiveness in precipitating cartilage matrix proteoglycans in situ. Dyes were introduced into media at the onset of processing and were present throughout both aldehyde fixation and osmium tetroxide post-fixation. Contrary to expectation, most of the dye-proteoglycan complexes generated and stable under aldehyde fixation conditions were found to be unstable during post- fixation despite the continuing presence of the dye. A similar phenomenon was also found for the cationic dyes commonly used for precipitation of proteoglycans in cartilage tissue sections (such as Acridine Orange, Alcian Blue, Azure A, Methylene Blue, and Ruthenium Red). Only two dyes, i.e, RHT and the newly tested RHT-CC, formed proteoglycan precipitates sufficiently stable to resist disruption and extraction during osmium tetroxide post- fixation. The latter may be particularly useful in semiquantitative analyses of proteoglycan content in unstained tissue sections owing to its intense brown-black color. For applications in which the osmium tetroxide post- fixation step may be omitted, TRC and PRT may also be valuable for semiquantitative histochemistry by virtue of their stable fluorescence and intense violet color signals, respectively.