An automatic analysis method for in situ hybridization using high-resolution image analysis

Abstract

Specific mRNA for alpha 1 (I) collagen has been detected on a cellular level by in situ hybridization using radioactively labelled alpha 1 (I) antisense RNA probes. We here present an automatic, quantitative, evaluation technique for the determination of grain densities using the high-resolution image analysis system IPS KONTRON. The reliability and objectivity of this method were evaluated by comparing the values of grains per cell obtained by conventional and automatic techniques following in situ hybridization with alpha 1 (I) collagen DNA probes in various specimens. The correlation coefficients between conventional and automatic analysis of grain densities were 0.97 for fibroblasts embedded into a three-dimensional collagen gel, 0.94 for dermal fibroblasts in skin obtained from a patient with progressive systemic scleroderma, and 0.90 for fibroblasts in a 2-week-old scar. All correlation coefficients were significant on the P less than 0.0001 level. This new analysis technique therefore allows a more rapid and reliable quantitative evaluation of in situ hybridization and may also be helpful in differentiating between various cell populations characterized by different biosynthetic capacities.