Combined use of in situ hybridization and unlabeled antibody peroxidase anti-peroxidase methods: simultaneous detection of type I procollagen mRNAs and factor VIII-related antigen epitopes in keloid tissue.

In this study, we developed methodology that allows the combined use of in situ hybridization and peroxidase anti-peroxidase techniques on the same tissue section. A human pro alpha 1(I) collagen cDNA and antibodies to factor VIII-related antigen were used on keloid tissue sections as a model for a fibrotic reaction. The basic protocols of the techniques were modified to obtain optimal results. The feasibility of this new method was demonstrated by elucidation of type I procollagen gene expression in the cells of blood vessel wall and the adjacent fibroblasts. In the case of capillaries, pro alpha 1(I) collagen mRNAs were detected within endothelial cells identified by the presence of factor VIII-related antigen. Pro alpha 1(I) collagen mRNAs were also found in close proximity of medium-size blood vessels, but in this context clearly outside the vessel wall. These results may contribute to the understanding of pathogenetic aspects of keloids and other fibrotic conditions. Thus, the combination of in situ hybridization and peroxidase anti-peroxidase techniques provides a useful tool to examine gene expression simultaneously both at mRNA and protein levels in fibrotic tissues. This methodology is also applicable to a variety of other biologic and pathologic situations.