A DOUBLE-STAINING TECHNIQUE FOR DETECTION OF GROWTH-HORMONE AND INSULIN-LIKE GROWTH FACTOR-I BINDING TO RAT TIBIAL EPIPHYSEAL CHONDROCYTES

In the present study a double-staining technique was developed to inve stigate simultaneous GH and insulin-like growth factor-I (IGF-I) bindi ng to chondrocytes in a monolayer cell culture. Rat tibial epiphyseal chondrocytes were isolated by enzymatic digestion and cultured in mono layer. GH and IGF-I were labelled with biotin. The affinity of the bio tin-labelled ligands was compared with unlabelled ligands in a radiore ceptor assay. To study the distribution of GH and IGF-I binding in the monolayer, chondrocytes were incubated with biotinylated ligands with or without an excess of unlabelled ligands, followed by incubation wi th Vectastain ABC complex, which was then reacted with diaminobenzidin e (DAB). Double staining was accomplished by carrying out the first re action with DAB in the presence of nickel ammonium sulphate to give a black precipitate, followed by incubation with the second ligand, then ABC complex and finally DAB in the absence of nickel ammonium sulphat e to give a brown stain. The presence of type-II collagen was demonstr ated by immunohistochemistry and used as a marker for differentiated c hondrocytes. Biotin-labelled GH and biotin-labelled IGF-I exhibited do se-dependent displacements of I-125-labelled GH and I-125-labelled IGF -I respectively from the chondrocytes in a radioreceptor assay. The di splacement curves were identical to those of unlabelled ligands indica ting that the affinity was unaltered. Binding of biotinylated GH to ce lls was seen throughout the culture in regions where there was little or no type-II collagen staining. IGF-I binding was predominantly local ized to cells at high density; areas which also showed a high degree o f staining for type-II collagen. The different locations of binding su ggest that epiphyseal chondrocytes in monolayer culture comprise a het erogeneous cell population and that IGF-I and GH have different target cells.