Application of the silver-gold intensified 3,3′ -diaminobenzidine chromogen to the light and electron microscopic detection of the luteinizing hormone-releasing hormone system of the rat brain

Abstract A highly sensitive, recently developed immunohistological method is introduced in the present study with special emphasis on the luteinizing hormone-releasing hormone system of the rat brain. The method utilizes the specific capability of the diammobenzidine endproduct, a frequently used chromogen in immunocytochemistry, to produce and bind silver grains from a special physical developer, following suppression of the argyrophilia of the nervous tissue by thioglycolic acid. Metal deposition into the immunolabelled structures results in a real Golgi-like appearance of immunoreactive profiles. Specificity of this silver method was confirmed by ultrastructural analysis, which showed that unlabelled elements did not bind silver. Using this method, more immunoreactive neurons and fibres were visualized than compared with the results of the traditional peroxidase-antiperoxidase method. The luteinizing hormone-releasing hormone-synthesizing neurons proved to be fusiform, exhibiting either smooth or rough surfaced contour. Unlabelled terminals established axo-somatic synapses on labelled perikarya. The juxtaposition of immunoreactive luteinizing hormone-releasing hormone profiles suggest the possibility of self-regulation within the luteinizing hormone-releasing hormone system. The main advantages of the method are the increased sensitivity with preserved selectivity and wide applicability in different fields of neuroscience (peptide and transmitter immunocytochemistry, double labelling, horseradish peroxidase tract tracing, X-ray analysis).