The Protein Man's Blog | A Discussion of Protein Research

Immunocytochemistry is one of the most commonly used methods for protein localization in the cells or tissues using specific antibodies. The overall credibility of the results from Immunocytochemistry depends on two factors:

• Antibody specificity: Monoclonal or Polyclonal antibodies can provide non-specific staining by binding with off-target proteins. To determine the specificity, immunoblotting or immunoprecipitation can be used. If the results do not show proper binding or non-specific staining, then this antibody must not be chosen for staining.
• Method Used for Immunostaining

Choosing a correct control is an essential aspect of biological research to enhance the chances of reproducibility of the data, which becomes a problem when a single antigen can provide multiple results and the controls are not coherent.

Controls can be crudely divided into two broad categories which will be further elaborated here:

Antigen controls

This group is dependent on the function of the controls used from the antigen perspective and its differential usage in the methods.

1. Positive Control: These controls are the samples that have the target protein in a known location such as specific cell type or subcellular compartment. Also, these controls should provide a fluorochrome or chromogenic-based visualization. To start with, the deduction of the positive controls can be taken on evidences based on previous experiments such as overexpression or knock out studies, interaction studies or fractional western blotting. Also, it should be clearly demonstrated that the antibody does not cross-react with the closely related proteins. However, positive controls can be a little misunderstood if the antibody is developed against an overtly (or ubiquitously) expressed or expressed at really low levels.
2. Negative Control: Negative controls are equally important for the validation of immunostaining. The priority aim of these controls is to validate that the result is only because of the interaction of the paratope of the antibody with the epitope of the targeted protein. This step is absolutely critical for both commercially purchased or self-developed antibodies. This control can also be demonstrated by using western blot analysis; however, immunostaining is most preferred. This step also reveals the presence of isoforms or physically associated proteins if non-specific staining is observed.
3. Endogenous tissue background control: Certain cell types or tissues have inherent autofluorescence or background color properties, which can be translated into misinterpretation of the results. To avoid these issues, the cells and tissues have "no primary antibody" and "no secondary antibody" controls, which should be inspected under fluorescent or bright-field illumination.

 Reagent Specificity Controls

1. No Primary Antibody Control: Here, the tissue is exposed only with the antibody diluent buffer without containing the primary antibody followed by the rest of the steps of the protocol. Staining detection in the tissues refers to non-specific binding of the secondary reagents or non-specific signals from the tissue itself which can further be deconvoluted using other controls.
2. No Secondary Antibody Control: To understand the non-specific staining of the primary antibody and secondary antibody, this step is necessary. The tissue is incubated with all the reagents except the secondary antibody.
3. Isotype Control: This control is used when monoclonal antibodies are utilized. The tissues are exposed with only antibody diluent (without having primary antibody) consisting a non-immune immunoglobulin of the same isotype such as IgG, IgG2, IgG, or IgM in the same concentration as that of the used primary monoclonal antibody. Rest of the procedure should be followed unmodified.
   This control is essential in validating the specificity of staining and confirms that the signals are not because of the non-specific interaction of the immunoglobulin molecules with the tissue. There should be no or negligible signal detection. 
4. Absorption Control: Absorption or preabsorption control is a worthwhile approach to validate the specificity of the antibody and the signal. Here, the antibody is mixed with a peptide used for generating the antibody, resulting in completely eliminating the probability of binding of the antibody with the proteins present in the tissue. Ideally, the tissue and the antibody have to be preincubated with the peptide individually to avoid non-specific epitope detection. The approach is very much similar to that of a positive control. However, this control can be a little ambiguous as many proteins are not adequately free of contaminations to make preabsorption works.