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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, 1 against a nuclear antigen (Ki-67), 1 against a cytoplasmic antigen (cytokeratin), and 1 against a cytoplasmic and membrane-associated antigen and a polyclonal antibody against a nuclear and cytoplasmic antigen (S-100) 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.