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, 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.
Vosse
Bettine A H
BA
Faculty of Medicine, University of Maastricht, Maastricht, The Netherlands.
Seelentag
Walter
W
Bachmann
Astrid
A
Bosman
Fred T
FT
Yan
Pu
P
D000911:Antibodies, Monoclonal; D001360:Avidin; D001710:Biotin; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods* / Q000592:standards; D009195:Peroxidase; D011933:Reagent Kits, Diagnostic / Q000592:standards*; D013194:Staining and Labeling; D016707:Tissue Fixation
D000911:Antibodies, Monoclonal; D011933:Reagent Kits, Diagnostic; D001360:Avidin; D001710:Biotin; D009195:Peroxidase