The importance of IHC / ICC controls
Appropriate controls are essential for the accurate interpretation of IHC / ICC results. A successful IHC / ICC experimental design produces results that demonstrate that the antigen is localized to the correct specialized tissues, cell types, or subcellular location. Optimizing the fixation, blocking, antibody incubation, and antigen retrieval steps will generate a strong and specific signal.
However, IHC / ICC experiments must include positive and negative controls to support the validity of staining and to identify experimental artifacts. Additionally, variations in antibody specificity, experimental conditions, biological conditions between tissue types, and even researchers can lead to inconsistent stains and lead to inaccurate conclusions. To facilitate consistent performance, detailed record-keeping is an important factor for IHC / ICC studies. Here, we describe several established controls that can be used to support the specificity of your IHC / ICC results.
Background control of endogenous tissues
Certain cells and tissues may have inherent biological properties that result in background staining that could lead to misinterpretation of the results. Before applying the primary antibodies, cells and tissues should be inspected under the microscope using fluorescence (for fluorescent labels) or bright-field (for chromogenic labels) illumination to ensure that there is no signal inherent in the tissue itself. For example, lipofuscin is an endogenous autofluorescent pigment that can be confused with positive staining.
No primary antibody control
Control is always necessary for which the tissue was incubated with Antibody Diluent, without the primary antibody included. This is followed by incubation with secondary antibodies and detection reagents. Staining with detection reagents alone should be negligible to the extent that it does not obscure the specific staining or resemble the specific staining pattern.
This control can be used when working with monoclonal primary antibodies. The sample is incubated with antibody diluent, supplemented with a non-immune immunoglobulin of the same isotype (eg, IgG1, IgG2A, IgG2B, IgM) and concentration as the primary monoclonal antibody. The sample is then incubated with the secondary antibody and detection reagents. These steps will help ensure that what appears to be specific staining was not caused by non-specific interactions of immunoglobulin molecules with the sample. Background staining should be insignificant and not resemble a specific stain.
To demonstrate that an antibody specifically binds to the antigen of interest, it is first preincubated with the immunogen. This should inactivate the antibody and the tissue should show little or no staining. The antigen and antibody mixture should be prepared at a 10: 1 working dilution (molar ratio) and pre-incubated overnight at 4 ° C. The pre-absorbed antibody can then be incubated with tissue instead of the primary antibody alone. The staining pattern produced by the primary antibody can be compared to that produced by the pre-absorbed antibody.
Absorption controls work best if the immunogens are peptides. However, if antibodies were raised against the entire protein, the addition of the antibody plus protein mixture may result in increased nonspecific staining. Although the mechanism is unclear, the antigen used for pre-absorption can bind to tissue and result in non-specific staining. Therefore, it is important to note that an absorption control using whole protein cannot always confirm the specificity of an antibody for the protein in the tissue.
Control of tissue type
Additional controls for IHC / ICC experiments include the use of tissue samples that are known to express (or do not express) the epitope of interest. This strategy can provide a useful reference and can also be used during initial optimization studies. Tissue from transgenic animals that overexpress or do not express antigen can be particularly useful. In addition, tissue samples from different species can be included to support the species specificity of an antibody.
Limitations of Western Blot Comparisons
Immunoblotting experiments are often performed to complement and support IHC / ICC studies. However, changes in protein conformation during denaturation can lead to potentially misleading results. For a given antibody, inconsistent results between Western blot and IHC / ICC studies may simply reflect differences in the experimental conditions employed. Since multiple epitopes are recognized, polyclonal antibodies are less vulnerable to such experimental artifacts.