Optimal Conditions for Detection of Calcitonin mRNA by In Situ Hybridization (ISH) Method Using a Non-radioactive Probe in the Rat Thyroid Gland

Abstract

Optimal conditions for detection of calcitonin mRNA were examined by in situ hybridization (ISH) method using a non-radioactive probe in the rat thyroid gland. An oligonucleotide complementary to rat calcitonin mRNA was synthesized with a DNA synthesizer, labelled at 3'-end by using digoxigenin-11-deoxyuridine triphosphate (dUTP) and terminal transferase (Boehringer Mannheim), and used as a probe. Hybrid chains formed by probe and calcitonin mRNA were visualized by anti-digoxigenin-alkaline phosphatase conjugate, nitroblue tetrazolium (NBT) and X-phosphate (Boehringer Mannheim) . To determine the optimal conditions for ISH, relations between tissue fixation and proteinase K treatment, and between hybridization temperature and time were mainly surveyed in the present study. As for the relation between tissue fixation and proteinase K treatment, good results were obtained in sections fixed by immersion in 10% formalin at 4°C for 2 hr, and digested with 1 μg/ml proteinase K at 37°C for 20 min. Under this condition, the most intense signals were obtained after hybridization at 37°C overnight. Alternative application of ISH and immunostaining to each of the adjacent sections revealed a small number of cells which were immunonegative but displayed hybridization signals.