Combined non-isotopic in situ hybridisation and indirect immunohistochemical analysis of hormone production in the rat pituitary gland.

Abstract

An understanding of the intracellular relation between hormonal expression (storage) and gene expression (production) is essential for elucidating the functional status of the individual cells in endocrine tissue such as the pituitary gland. To this end, mRNA expression was visualised by using a combined in situ hybridisation and immunohistochemistry method in routinely processed, formalin fixed, paraffin wax embedded rat pituitaries. mRNA was detected by non-isotopic in situ hybridisation (alkaline phosphatase antialkaline phosphatase method, with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate as substrates). Sections were then stained by using the immunoperoxidase method to demonstrate pituitary hormone expression. The specificity of the combined staining method was confirmed by staining adjacent sections separately. The antigenicity of rat growth hormone and prolactin was adequately preserved following hybridisation. In conclusion, this method is specific, easy to use and permits the determination of the functional status of individual cells.