STUDIES ON THE PHYSICAL DEVELOPER FOR USE IN IMMUNOHISTOCHEMISTRY

Four experiments were carried out to investigate physical developers containin In experiment 1, the effect of citric acid concentrations in physical developers upon the intensity of immunohistochemical reactions was investigated, using a protein A-gold silver technique for detection of insulin. It was noted that the reactions were decreased in intensity with increasing concentrations (300, 400, 500 and 700 mg/61 ml) of citric acid involved. Addition of 250 mg/61 ml sodium citrate to the developer containing 700 mg/61 ml citric acid elevated pH to a value nearly comparable to that of the developer containing 300 mg/61 ml citric acid. Such a maneuver increased the intensity of the reaction, however, not to a level reached when the developer containing 300 mg/61 ml citric acid was used. Thus, it is considered that citric acid controls the developing capacity not only by modulating pH values but by complexing with silver. In experiment 2, the possibility was substantiated that the citric acid involved can be replaced with maleic acid. In experiments 1 and 2, we newly prescribed two refined physical developers (Developers 2 and 3). Developer 2 contains a larger amount (500 mg/61 ml) of citric acid as compared with Developer 1 which we have thus far employed primarily. In Developer 3, maleic acid is contained instead of citric acid. In experiment 3, these revised physical developers were used in protein A-gold silver or immunogold silver staining for detection of a series of antigenic substances, and proved to be excellent, in terms of the intensity, sensitivity and specificity of histochemical reaction. Further, experiment 4 in which Danscher's physical developer was tentatively used in immunostaining substantiated the superiority of the present revised developers to the one elaborated by Danscher (7).