Controls for immunocytochemistry: an update

Abstract

Immunocytochemistry is a highly productive method in biomedical research used to identify proteins and other macromolecules in tissues and cells. Control samples are required to show label localization is correct, but the understanding and use of immunocytochemistry controls have been inconsistent. A new classification of immunocytochemical controls is proposed that will help in understanding this most important component of the experiment. The three types of controls required for immunocytochemistry are primary antibody controls that show the specificity of the primary antibody binding to the antigen, secondary antibody controls that show the label is specific to the primary antibody, and label controls that show the labeling is the result of the label added and not the result of endogenous labeling. Publications containing immunocytochemical results must give details of how these controls were performed.

Figure 1.

An immunoglobulin G (IgG) antibody is a single protein made from four peptides joined by disulfide bonds. There is a single constant region (white) containing the Fc portion and species-specific antigens. The variable region (gray) contains the Fab portion that binds the epitope portion of the antigen. The short protein found only in the variable region is known as the light chain; the large protein that is part of the constant and variable region is the heavy chain. The IgG can be digested by the protease enzyme, papain, at the hinge region (flexible region of the heavy chain), into an Fc end (constant end) and a Fab end (variable end). The antigen is the molecule used to immunize the animal, and the epitope is one of many portions of the antigen that can generate antibodies. Figure reproduced with permission from Burry (2010).

Figure 2.

Absorption control is the incubation of the primary antibody with the antigen used to generate the antibody. (A) The primary antibody incubated with excess antigens binds all of the Fab sites capable of binding the antigen in the tissue (arrow). (B) If the correct antigen and an incorrect antigen have the same epitope (arrows), then binding to both is inhibited by the absorption control. (C) In some cases, the antigen adsorbed by the antibody binds to proteins in the tissue, and the adsorbed antibody appears to bind to a protein (#4) independently of the antibody. Figure reproduced with permission from Burry (2010).

Figure 3.

(A) Incubation with antibodies should show antibody binding to only the correct antigen. Nonspecific binding can result from charged groups that bind proteins, including antibodies. The tissue can have Fc receptors that bind to the Fc region of any antibody. Some tissues can have exposed endogenous antibodies. In experiments with multiple primary antibodies, incorrect binding by other antibodies can occur. (B) There are blocking agents that block each of the sites that cause nonspecific binding. The primary antibody binds to the correct antigen and is not affected by any of the blocking agents. Charged groups can be quenched by any protein, and BSA is commonly used because it is not a source of antibody binding. The Fc receptors must be quenched by the Fc end of an IgG antibody that has no ability to bind other antigens; normal serum from the same species as the secondary antibody is commonly used. Endogenous antibodies are blocked by incubations with antispecies Fab fragments that are used only when tissue from injured animals is processed. In experiments with multiple primary antibodies, where incorrect binding of labeled secondary antibodies is suspected, the secondary antibody control shows this incorrect binding. Reproduced with permission from Burry (2010).