Improved tissue preparation for in situ localization of specific mRNA using non-radioactive DNA probes: Effects of protease digestion and probe size on signal detection in frozen and paraffin sections of rat pituitary glands.

Abstract

To better describe the physiological state of cells, detection of specific mRNA by in situ hybridization at the cellular level is often demanded. In order to accomplish this reliably, various factors such as morphological preservation, retention of mRNA, accessibility of the labeled probe to the target mRNA and efficency of the hybridization should be considered during the tissue processing. In this paper, using non-radioactive probes, we investigated the effects of protease treatment and the size of probe on the efficiency of in situ hybridization with mRNAs in frozen and paraffin-embedded tissue sections of the rat pituitary glands. As non-radioactive haptenic DNA probes, we used dinitrophenyl (DNP)-labeled proopiomelanocortin (POMC) DNA and thymine-thymine (T-T) dimerized prolactin cDNA. The signals were visualized by the indirect enzyme-immunohistochemistry. The best results were obtained when frozen sections of tissues fixed by 4% paraformaldehyde in phosphate buffered saline were mildly digested with protease and hybridized with the haptenized DNAs of about 200-400 base pairs.