Abstract
Immunohistochemistry (IHC) is a method of routine use in histopathology for the diagnosis and differentiated diagnosis of liver diseases. IHC staining in human hepatic tissue is often obstructed by the presence of non-specific antibody interactions, the cause of high background staining. Cognizant of previous encounters with high levels of background in human hepatic tissue when using polyclonal antibodies, the potential causes were systemattically investigated by testing the impact of different antibody variants and assay conditions. Firstly, IHC was performed on hepatic tissue with immunized and non-immunized polyclonal antibodies and it was demonstrated that immunized antibodies pervaded the samples with non-specific staining, while non-immunized antibodies produced insignificant background. Following, the primary antibody was co-incubated with different concentrations of protein blocks, acid/base solutions, crystalline ionic compounds, and combinations of these individual components in order to assess their potency in removing background staining. Optimization of these assay conditions revealed that background could be significantly reduced by the addition of sodium chloride to primary antibody solution and/or protein block to sodium azide-stabilized Tris-HCl buffer, while little beneficial effect was observed from co-incubating the primary antibody with non-serum protein block/goat serum, hydrochloric acid/sodium hydroxide, or a higher concentration of peroxidase block. Together, the results suggest that hepatic background in IHC is mainly attributed to strong ionic interactions between the light chains of polyclonal antibodies and polar molecules specifically prevalent in hepatic tissue, which can be reduced by increasing the ionic strength of the antibody–antigen reaction microenvironment. Thus, the findings can enhance the specificity of hepatic tissue immunoassays and improve IHC studies in human liver tissues using polyclonal antibodies.
Published Version
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