Immunohistochemistry (IHC) & Antigen/Epitope Protein Retrieval - Principle, Technique and Protocol

Overview of Immunohistochemistry (IHC)

Diagnostic pathology has exponentially advanced over the last few decades due to the implementation of various novel techniques. The most notable of these techniques for diagnosis is immunohistochemistry (IHC). BioGenex, a pioneer in molecular pathology solutions and precision medicine, has been serving the diagnostic community for over 30 years and has been known for the continued development of innovative IHC products.

What is Immunohistochemistry?
Immunohistochemistry is a well-established, highly sensitive method that allows the localization of an antigen within a cell or a tissue with high resolution. IHC technique is based on a primary antibody that specifically binds to its complementary antigen. The bound antigen-antibody complexes are then visualized using chemically linked enzymes, dyes, or fluorescent labels.  The method combines molecular detection with morphological features and can be used on formalin-fixed paraffin-embedded (FFPE) sections, frozen sections, and other cytological preparations. IHC is also used for the elucidation of differential diagnoses, a limitation for conventional hematoxylin and eosin analysis. The breakthrough developments of a) monoclonal antibodies, b) antigen exposure technique in preserved tissues, c) sensitive visualization methods, and d) automated strainers have made IHC an indispensable tool for modern molecular diagnostic laboratories.

Immunohistochemistry use in diagnostics and research

Immunohistochemistry(IHC) ProtocolFig. 1 Protocol for Immunohistochemistry (IHC)

IHC markers are specific molecular signatures characteristic of cellular events, and changes in expression level are often associated with abnormal cellular events like proliferation and apoptosis. The unique biomarkers are targeted for identification and detection in IHC assays for diagnostic use.

IHC use in cancer diagnostics involves differentiation of benign and malignant tumors, determination of the cell lineage of undifferentiated malignant tumors, differentiation of tumor subtypes, prognostic markers for a suitable therapeutic regimen, and infectious agent identification.

In breast cancer, IHC is used for differentiating invasive breast carcinoma from non-invasive lesions by using basal layer markers, like p63. The primary site of tumor origin is identified in cases of metastatic carcinoma by using classification markers like cytokeratins. Expression of certain biomarkers are identified using IHC for determination of specific treatment strategies, the best example is trastuzumab (Herceptin®) treatment for breast cancer patients expressing HER2 receptors.

Disease prognosis is another clinical application of IHC where the likely course of the disease is studied, for example; the expression of cell proliferation marker Ki-67 is monitored to predict the patient survival in urological cancers.

IHC in research applications includes antitumor drug development and biomarker discovery. IHC helps to investigate the expression of specific proteins and co-localization of proteins within the membrane, cytoplasm, or nucleus to study the altered expression in normal and abnormal cells.

Immunohistochemistry procedure:

 The IHC staining procedure covers from sample collection to final visualization and involves section preparation, antigen retrieval, immunostaining, and detection.

Proper preparation of tissue samples is critical for optimal detection. Tissue samples are obtained from in vivo host biopsies and are rapidly preserved to prevent hypoxia, lysosomal enzymes, and putrefactive changes due to bacterial and mold growth. The preservation step maintains tissue architecture, prevents degradation of proteins, and maintains the antigenicity of the targeted probe.   

Fixation of tissues

Tissue fixation uses fixatives like 10% neutral buffer formalin (NBF), formaldehyde, paraformaldehyde, or formaldehyde-free solutions. The fixation retains the tissue morphology, cell structure and position, prevents diffusion of proteins, and inhibits endo- and exogenic enzymatic degradation. The FFPE tissue sections are sectioned into thin sections of 3-7 µm or less using a microtome. These sections are the most common form of tissue samples used in IHC.

Antigen retrieval

Fixative agents fix tissue by forming methylene bridges (cross-linking of amino acids) within the protein structure or with other proteins, often leading to the masking of antigenic epitopes. This results in weak to negative staining, even for highly expressed antigens. In 1991 BioGenex invented the ‘Antigen Retrieval’ technique (1) to unmask antigenic epitopes by boiling the FFPE tissues section in a buffer under controlled temperature conditions. Using the EZ Retriever® Microwave System large collections of archived FFPE tissue sections that were available in every pathology lab could be turned into valuable medical samples for diagnostics and translational research. Boiling FFPE tissue sections in buffer break the aldehyde bonds formed during the fixation and helps in recovering the masked epitopes.

BioGenex has developed retrieval solutions for antigen retrieval, decal retrieval in decalcified tissues and nucleic acid retrieval for in situ hybridization (ISH) applications. BioGenex’s new EZ-AR™ Elegance series of retrieval solutions eliminates common issues like improper heat distribution, buffer evaporation, tissue drying, and boil overs during antigen retrieval. EZ-AR Elegance solutions with EZ Retriever System streamline the retrieval process including deparaffinization, rehydration, and antigen retrieval.

Blocking nonspecific staining

Blocking agents help eliminate nonspecific background staining. Endogenous enzyme activity will cause high, non-specific background staining during IHC assays. Endogenous enzymes like peroxidase and phosphatase are blocked by saturating the tissue sections with an appropriate blocking agent, like hydrogen peroxide, acetic acid, or levamisole. Non-specific antibody binding is prevented using sera, BSA, or casein protein. In special cases, biotin blocking may be required for the usage of the biotin-based detection systems.

BioGenex caters blocking agents for both types of non-specific staining i.e., Peroxide Block for blocking endogenous enzymes and Power Block™ for blocking non-specific antibody binding. BioGenex Power Block™ is a universal blocking agent used to reduce non-specific antibody binding and also works as antibody diluent for concentrated antibodies. 

Immunostaining

Immunostaining uses antibodies to detect the distribution and localization of the specific antigen of interest. IHC markers specific to disease conditions are chosen based on the tissue type and clinical relevance, along with proper control tissues. Clinical information of the antigen like its function, subcellular localization, and expression profile helps in antibody selection. The clonality of the antibodies, polyclonal vs monoclonal, is determined based on the assay specificity. Polyclonal antibodies recognize multiple epitopes of the same antigen, enhancing the signal. Polyclonal antibodies are more resistant to conformational changes and are used in assays that requires high sensitivity. Monoclonal antibodies are used for high specificity assays as they bind to a single epitope, exhibit low cross-reactivity, and minimal background.

BioGenex offers over 400+ primary antibodies for cancer diagnosis that include monoclonal and polyclonal antibodies from mouse or rabbit clones. All BioGenex antibodies are tested and validated for IHC on human FFPE tissues for both manual and automated staining using Xmatrx® systems.  BioGenex antibodies come in different formats and volumes to accommodate manual (ready-to-use dropper bottles) and automated (barcoded vials) staining procedures. Most of our antibodies are IVD certified and CE-marked and a few are available for RUO. Our antibodies also come in the concentrated format along with the Enhanced Antibody Diluent for dilution of all antibodies to improve antigen-antibody interaction and affinity and to reduce antibody loss due to adsorption.

Detection methods

Primary antibody detection within an IHC experiment can be done using either direct or indirect assays. In a direct assay, the primary antibody is covalently tagged to a label to detect highly expressed antigens. This method is commonly used for multiplexing with multiple antigen targets. Indirect detection relies on the utilization of a labeled secondary antibody containing reporting enzymes for signal amplification. The target antigen for both methods is visualized using a fluorophore or chromogenic substance.

BioGenex Detection Systems are high sensitive kits that give a high-resolution visualization of the bound antibodies with a variety of colorimetric endpoints. Our Super Sensitive™ (SS) Polymer-HRP IHC Detection System uses a novel polymeric technology universal for both rabbit and mouse antibodies, offers excellent cell diffusion for intense nuclear, cytoplasmic and membrane staining. Horseradish peroxide linked to a polymer chain enhances the signal by catalyzing higher molar ratios of the substrates giving intense stains. The kit is non-biotin based to eliminate the cross-reactivity with endogenous biotin to give clean background. The Super Sensitive™ One-Step Polymer HRP IHC Detection System offers a fast 15 minutes staining with direct visualization after antibody binding. The Super Sensitive™ Link-Label IHC Detection System is for biotin based detection systems that use streptavidin-biotin interaction to detect bound antibodies. This uses a species-specific secondary antibody conjugated to biotin. The bound secondary antibody then reacts with the streptavidin conjugated with an enzyme which on reaction with a suitable substrate forms a colored reaction. BioGenex also offers different chromogens that include DAB, AEC, Elegance Red, Fast Red, and New Fuchsin available for both manual and automated staining protocols.

Related techniques
In situ hybridization, Fluorescent in situ hybridization

References

1: Shi SR, Key ME, Kalra KL. Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem. 1991 Jun;39(6):741-8. PubMed PMID: 1709656.