ICC/IHC Protocols

Protocol for VisUCyte™ HRP Polymer Detection Reagent

VisUCyte HRP Polymer is a biotin-free detection reagent which overcomes problems of avidin-biotin detection chemistry, such as endogenous biotin staining and the need to do additional quenching of endogenous biotin and avidin present in some tissues. With VisUCyte HRP Polymer it is possible to achieve specific staining in cells and tissue much faster than the conventional avidin-biotin-HRP procedure.

Detection & Visualization of Antibody Binding

Following incubation with the primary antibody, antibody binding is visualized using an appropriate detection system. The method of detection can be direct or indirect, and may generate a fluorescent or chromogenic signal. Direct detection involves the use of primary antibodies that are directly conjugated to a label. Indirect detection methods utilize a labeled secondary antibody raised against the primary antibody host species. Indirect methods can also include amplification steps to increase signal intensity.

Designing a Successful IHC/ICC Experiment

Immunohistochemistry (IHC) and immunocytochemistry (ICC) are techniques employed to localize antigen expression and are dependent on specific epitope-antibody interactions. IHC refers to the use of tissue sections, whereas ICC describes the use of cultured cells or cell suspensions. In both methods, positive staining is visualized using a molecular label, which can be fluorescent or chromogenic. Briefly, samples are fixed to preserve cellular integrity, and then subjected to incubation with blocking reagents to prevent non-specific binding of the antibodies.

Preparing Samples for IHC/ICC Experiments

Tissue and cell samples must be appropriately harvested and prepared for each IHC/ICC study. To facilitate the required incubation steps, whole tissues must be cut into ultra thin (5-10 µm) sections or cut into smaller pieces for whole mount IHC. For ICC experiments, cells must be attached to a microscope slide or coverslip before commencing the staining procedure. Sample preparation is also intimately linked to the method of fixation, which in turn is influenced by the desired detection technique (fluorescence versus chromogenic).

Preventing Non-specific Staining

Once tissue or cell samples have been appropriately prepared and fixed, the samples are ready to be stained. All IHC/ICC studies are dependent on specific antibody-epitope binding, which is governed by hydrophobic interactions, ionic interactions, hydrogen bonding, and other intermolecular forces. However, the same attractive forces can also result in non-specific staining, i.e. binding of the primary antibody to amino acids other than those within the desired epitope of the antigen. This is a common problem that occurs in IHC/ICC experiments.

The Importance of IHC/ICC Controls

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.

Protocol for Heat-Induced Epitope Retrieval (HIER)

Antigen retrieval can reveal epitopes masked during the preparation of tissues for staining. The protocol below describes a technique used by R&D Systems and can be used on both cryostat and paraffin-embedded sections. The protocol can be used with R&D Systems Acidic, Basic, or Universal Antigen Retrieval Reagents. Pretreatment with these solutions may induce a dramatic enhancement of immunoreactivity. However, they also have the potential to affect tissue morphology.

Appropriate Fixation of IHC/ICC Samples

All samples used in IHC/ICC experiments must be fixed to preserve tissue morphology and retain the antigenicity of the target molecules. Fixation alters the chemical composition of tissues and often requires a compromise between preserving tissue structure and preserving the epitope. Incomplete fixation (underfixation) of cells or tissues may allow rapid proteolytic degradation of target proteins within the tissue and can reduce specific immunoreactivity.

Antigen Retrieval Methods

Although fixation is essential for the preservation of tissue morphology, this process can also have a negative impact on IHC/ICC detection. Fixation can alter protein biochemistry such that the epitope of interest is masked and can no longer bind to the primary antibody. Masking of the epitope can be caused by cross-linking of amino acids within the epitope, cross-linking unrelated peptides at or near an epitope, altering the conformation of an epitope, or altering the electrostatic charge of the antigen.