APPLICATION AND COMPARISON OF SILVER INTENSIFICATION METHODS FOR THE DIAMINOBENZIDINE AND DIAMINOBENZIDINE NICKEL END-PRODUCT OF THE PEROXIDATION REACTION IN IMMUNOHISTOCHEMISTRY AND INSITU HYBRIDIZATION

Silver-intensification methods described in the literature for the dia minobenzidine (DAB) and diaminobenzidine-nickel (DAB/Ni) endproduct of the peroxidase reaction were compared in model systems after immunope roxidase and in situ hybridization. First, these methods were compared in immunohistochemical model systems, using the demonstration of glia l fibrillar acidic protein (GFAP) and prostate-specific antigen (PSA) in paraffin sections of human brain and prostate tissue, respectively. When DAB without Ni was used as substrate, tissue argyrophilia caused considerable background staining. Only when this tissue reactivity wa s quenched with, e.g., CuSO4 with H2O2 or thioglycolic acid, were the results acceptable. A considerable improvement in the signal-to-noise ratio could be obtained when nickel was included in the substrate mixt ure. The methods that proved to be best for demonstration of GFAP and PSA made use of acid developer solutions. Subsequently, these methods were compared with other sensitive immunostaining methods for demonstr ation of the gamma-delta T-cell receptor in frozen lymphoid tissue. In this model a considerable increase in the number of positive cells co uld be obtained using silver intensification. The different methods us ing DAB/Ni were also compared for use in DNA in situ hybridization (DI SH). In this case two model systems were used: human papilloma virus t ype 11 (HPV-11) DNA in condyloma tissue (abundant target model) and Ep stein-Barr virus (EBV) DNA in a mononucleosis lymph node (low target m odel). For demonstration of HPV-11, all methods gave more or less sati sfactory results, which were best with the acid developer solutions. M oreover, for demonstration of EBV DNA, a signal could be obtained only with these developer solutions. Such a method also proved suitable in double immuno-hybrido stainings for the demonstration of EBV DNA in s pecific antigen-positive Reed-Sternberg cells in paraffin sections of Hodgkin lymph nodes.