Abstract

An improved protocol for in situ hybridization(ISH) to routinely processed, paraffin-embedded tissue sections from lung carcinoma is presented. For this study, DNA probes for alpha-satellite chromosome 7 and 17 were used. The protocol to detect numerical chromosome aberrations involved treatment of sections with 1 M sodium thiocyanate prior to pepsin digestion, resulting in reproducible ISH reactions. The effect of avidin-biotin detection system. Four layer avidin methods and triple biotin methods, using avidin-PO, goat antiavidin, biotinylated antigoat IgG, avidin-PO or anti-biotin, biotinylated antirabbit IgG, avidin-PO, markedly enhanced the intensity of positive signals. More than 80% of the tumor and stromal cells showed distinct chromosome hybridization signals in 6 micrometer-thick sections. Lung carcinoma cells showed multiple chromosome signals(2~5 spots), contrasted by one or two signals in the stromal cells in the same section. These results suggest that chromosome polysomy can be reliably detected in tissue sections using in situ hybridization. This capability will prove to be an important tool for determining the underlying genetic basis for tumor development, tissue phenotype heterogeneity and progression by allowing genetic determination to be made on paraffin-embedded tissue sections where tumor histologic architecture is preserved.

• Keywords: In situ hybridization;Chromosome aberration;Avidin;Biotin;DNA probes