Background staining of visualization systems in immunohistochemistry -: Comparison of the Avidin-Biotin complex system and the EnVision+ system

The aim of this study was to evaluate specific immunostaining and background staining in formalin-fixed, paraffin-embedded human tissues with the 2 most frequently used immunohistochemical detection systems, Avidin-Biotin-Peroxidase (ABC) and EnVision(+). A series of fixed tissues, including breast, colon, kidney, larynx, liver, lung, ovary, pancreas, prostate, stomach, and tonsil, was used in the study. Three monoclonal antibodies, I against a nuclear antigen (Ki-67), 1 against a cytoplasmic antigen (cytokeratin), and I against a cytoplasmic and membrane-associated antigen and a polyclonal antibody against a nuclear and cytoplasmic antigen (S100) were selected for these studies. When the ABC system was applied, immunostaining was performed with and without blocking of endogenous avidin-binding activity. The intensity of specific immunostaining and the percentage of stained cells were comparable for the 2 detection systems. The use of ABC caused widespread cytoplasmic and rare nuclear background staining in a variety of normal and tumor cells. A very strong background staining was observed in colon, gastric mucosa, liver, and kidney. Blocking avidin-binding capacity reduced background staining, but complete blocking was difficult to attain. With the EnVision(+) system no background staining occurred. Given the efficiency of the detection, equal for both systems or higher with EnVision, and the significant background problem with ABC, we advocate the routine use of the EnVision(+) system.