Appropriate controls are critical for the accurate interpretation of IHC/ICC results. A satisfactory IHC/ICC experimental design produces results that demonstrate that the antigen is localized to the correct specialized tissues, cell types, or subcellular location. Optimization of 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 identify experimental artefacts. In addition, variances in antibody specificity, experimental conditions, biological conditions between tissue types, and even researchers may generate inconsistent staining 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.
Endogenous Tissue Background Control
Certain cells and tissues may have inherent biological properties resulting in background staining that could lead to a misinterpretation of the results. Before applying primary antibodies, cells and tissues should be inspected under the microscope using either ?uorescence (for fluorescent labels) or bright-field (for chromogenic labels) illumination to ensure there is no signal inherent to the tissue itself. For instance, lipofuscin is an endogenous autofluorescent pigment that can be confused with positive staining.
|Lipofuscin Background in Nervous System Tissue. Lipofuscin is a pigment that accumulates with age in many tissue types. It also has autofluorescent properties that overlap with excitation and emission spectra of commonly used fluorochromes. Circled in the micrographs above are lipofuscin-containing neurons that may appear labeled using either bright-field microscopy (A) or fluorescence microscopy in the green (B) and red spectrums (C).
No Primary Antibody Control
A control in which the tissue is incubated with antibody diluent, without the primary antibody included, is always necessary. This is followed by incubation with secondary antibodies and detection reagents. Staining with detection reagents alone should be negligible to the point that it does not obscure specific staining or resemble the specific staining pattern.
This control can be utilized when working with monoclonal primary antibodies. The sample is incubated with antibody diluent, supplemented with a non-immune immunoglobulin of the same isotype (for example, 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 negligible and not resemble specific staining.
To demonstrate that an antibody is binding specifically to the antigen of interest, it is first pre-incubated with the immunogen. This should inactivate the antibody and the tissue should show little or no staining. The antigen to antibody mixture should be made at a working dilution of 10:1 (molar ratio) and be pre-incubated overnight at 4 °C. The pre-absorbed antibody can then be incubated with tissue in place 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 better if the immunogens are peptides. However, if antibodies were raised against the whole protein, addition of the mixture of antibody plus protein may result in higher non-specific staining. Although the mechanism is unclear, the antigen used for pre-absorption may itself bind to the tissue and result in non-specific staining. Thus, it is important to note that an absorption control using whole protein may not always confirm the specificity of an antibody for the protein in the tissue.
|Absorption Control in Rat Dorsal Root Ganglion. A. A cryostat section of rat dorsal ganglion stained for phospho-MSK1 (S212) using anti-human phospho-MSK1 affinity-purified polyclonal antibody (Catalog # AF1036). B. Nuclear staining (indicated by arrows) is abolished if the antibody is first pre-absorbed with the S212 phosphorylated immunogen.
Tissue Type Control
Additional controls for IHC/ICC experiments include using tissue samples that are known to express (or not express) the epitope of interest. This strategy can provide a useful reference and may also be utilized during initial optimization studies. Tissue from transgenic animals that overexpress or do not express the 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
Western blot experiments are often conducted to complement and support IHC/ICC studies. However, changes in protein conformation during denaturation can result in 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.