Constants and variables in immunohistochemistry

Many efforts have been made to develop reliable methods for the quantification of immunohistochemical reaction products. Most of the corresponding studies have dealt with problems related to the development of adequate hardware and software, while fewer investigations have focused on variables of histotechnical or immunohistochemical methods. The present paper summarizes findings and experience obtained over many years in this latter field, and a total of 14 corresponding parameters were considered. The studies were performed with methods well established in the author's laboratory; namely immunohistochemistry for various pancreatic hormones and chromogranin A applying the peroxidase anti-peroxidase method on serial semithin sections from the mammalian endocrine pancreas. Optical densities of immunoreactivities were determined using an appropriate measuring program by the interactive image analysis system IBAS. All parameters investigated were found to influence densities of immunoreactivities, and those with major significance were: 1) the thickness of histologic sections; 2) the dilution range of the antisera used as first layers; 3) the type or composition of the buffers used for dilution of the antisera and of the chromogen di-aminobenzidine or as the rinsing solution. All these variables could be standardized in appropriate ways. It was not possible, however, to prevent batch-to-batch (inter-assay) variations. Finally, the results of the present investigations served to increase the efficiency of immunohistochemical staining by the applied methods.