Non-specific background staining

Tissue was allowed to dry before fixing or during staining. 

Folds within the tissue section trapped reagents. 

Antigen migrated in tissue (common in frozen sections). 

Polyclonal antibodies that are not affinity purified may contain non-specific 

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Non-specific background staining may increase as the signal increases. 

The optimal dilution for use of an antibody in immuno-histology is defined as the dilution at which the greatest contrast is achieved between the desired (specific) positive staining and any unwanted (non-specific) background staining. Selection is subjective and is based not simply... 

Incubation of slides in a humidity chamber is advisable whatever the temperature. In practice, if a slide treated with antibody is permitted to dry, excessive non-specific background staining invariably occurs. Even when total drying does not occur, evaporation... 

FIGURE 1.24 Section of liver illustrating endogenous biotin resulting in non-specific background staining. 

EDTA and Tris/EDTA-based high pH buffers are useful for antibodies with low expression levels or when the tissue is over-fixed, but the tissue sections suffer under the high pH conditions and a non-specific background appears. EDTA or Tris/EDTA-based buffers are also commercially available under different names such as Target Retrieval Solution, pH 9 (Dako). Different observations reported non-specific nuclear staining using retrieval solutions containing Tris buffer, the addition of urea could solve this problem. 

The first step in immunochemical detection of proteins after electrotransfer is blocking the support with an inert material to inactivate further non-specific binding of protein. The blocking reagent should cover the membranes at those areas where no blotted protein is bound and should not react with any of the reactants of immunochemical detection cascade as indicated by no non-specific staining, i.e., resulting in blank background of the membrane. 

An unfortunate, widespread practice in EIA is the inclusion of a number of negative serum samples at a single dilution to establish mean and SD of background staining, whereas the test samples are serially diluted. The assumption that non-specific binding (observed with negative sera) is constant, irrespective of the dilution factor violates the law of mass action. Serial dilutions should, therefore, also be used with the negative sera. 

Mason and Sammons (1979) introduced an elegant method to lower background staining, caused by non-specific adsorption of IgG to tissue sections, by coupling the enzyme to purified antigen. An excess of the primary antibody is incubated with the tissue preparation, but among the IgG adsorbed only the antibodies reacting... 

A common problem with the indirect covalently labeled enzyme methods is that anti-Ig antibodies often adsorb non-specifically to the tissue, resulting in background staining (i) when long incubation or staining periods are used and, (ii) when the ratio of specifically bound and non-specifically adsorbed antibodies becomes smaller (sparsity of antigens or primary antibodies large excess of anti-Ig antibodies). 

Coomassie blue staining of proteins after their separation by electrophoresis is based on non-specific binding of Coomassie dye to proteins. The proteins are detected as blue spots on a clear background. 

Negative (non-specific) Patient tissue with components 1 that are the same as tissue to be studied Processed in same way as patient sample Diluent (as used with antibody) without antibody Antibody not specific for antigen of interest in same diluents as used with kit antibody Detection of unintended background staining... 

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