Cytochemical staining of connective tissue extracellular matrix with ruthenium hexammine trichloride (RHT)

Abstract

The extracellular matrix (ECM) of fibrous connective tissue is a composite of protein- and carbohydrate-containing structures. Preservation of the protein components is achieved through fixation with glutaraldehyde and osmium tetroxide. Carbohydrates however are inadequately preserved and often lost using these fixatives. Cationic dyes such as ruthenium red, alcian blue, and several others have been used to stabilize and retain carbohydrates, particularly the proteoglycans (PGs), but usually with less than optimal results. The present study documents the improved preservation and staining of PGs and other carbohydratecontaining components of skin ECM using ruthenium hexammine trichloride (RHT).Human neonatal foreskins were cut into 1 mm 3 blocks, fixed for 3 h in cacodylate buffered (0.1M, pH 7.2) 2.5% glutaraldehyde containing 1 mg/ml RHT (Polysciences, U.S.A.), postfixed 1 h in buffered 1% OsO4 also containing 1 mg/ml RHT, dehydrated in ethanol and embedded in Spurrs. In some cases the tissue was digested with chondroitinase ABC (Miles, U.S.A.) prior to fixation. Sections from tissue fixed without RHT were poststained in uranyl acetate and lead citrate. Figs. 2 - 5 are from 0.5 μm sections of tissue immunolabelled with monoclonal antibodies to type VI collagen.