Immunohistochemistry (IHC)

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Immunohistochemistry (IHC) is an antibody-based technique that detects antigens of interest in fixed tissue sections, often visualized by light microscopy (see figure below). In this multi-step application, successful staining is highly influenced by multiple variables. High-quality reagents and optimized IHC protocols are key for generating accurate expression, localization, and distribution data. Our complete line of IHC products and scientific resources will help you produce valid, reproducible results you can trust.

Traditional Chromogenic IHC Staining and Detection. As illustrated above, enzymatic conversion of a chromogen substrate is required to generate a signal and visualize an antigen in traditional IHC assays. Fluorescence detection is an alternative method that is more suitable for multicolor experiments due to the broad availability of fluorochromes and the emergence of high contrast imaging. Read more about IHC detection methods.

IHC Primary Antibodies

The most critical factor to achieve successful IHC staining is selecting an antibody that specifically binds the target antigen. Novus offers over 42,000 IHC validated antibodies referenced in more than 17,000 peer-reviewed publications that can be tested risk free with the Novus Quality Guarantee. Read more to learn about antibody selection and optimization for effective IHC staining.

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Secondary Antibodies and Detection Reagents

Frequently, IHC uses the indirect detection method in which a secondary antibody, directed against the constant region of the primary, carries the label (fluorescent, enzymatic, biotin, etc.) Both direct and indirect detection methods can be visualized by either immunofluorescence (IF) or by a chromogenic reaction. In IF detection, the fluorochrome conjugated antibody is excited by and emits light at specific wavelengths. In chromogenic detection, the antibody is conjugated to an enzyme (such as HRP) which converts DAB or 3-amino-9-ethylcarbazole (AEC) to a colored precipitate at the antigen site.

Compared to directly labeled primary antibodies, indirect detection is more sensitive and vital for effective identification of low abundance antigens, rare epitopes, and under conditions of nonoptimal antigen-antibody binding. Additionally, with the indirect method of detection multiple labeled secondary antibodies can bind to a single primary antibody. In some cases, such as for detection of low abundance antigens, additional steps to amplify the antigen signal may be required. Learn more about secondary antibodies.

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Tissue Samples and Controls

Due to the number of variables that impact staining, including the proper controls in IHC experiments such as tissue controls are important for identifying the source of staining issues and for validating results. Recommendations to verify the specificity of antigen-antibody interactions include absorption controls involving blocking peptides and isotype controls, which are used in place of primary antibodies.

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IHC Support Products

Novus further supports your IHC experiments by offering a wide range of supplementary reagents and complementary tools.

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Moving Beyond Protein Expression - Dual in situ Hybridization (ISH)/IHC

Combining the protein detection capabilities of IHC with single-molecule RNA expression analysis using RNAscope^® ISH technology builds a more complete and robust picture of the molecular mechanisms involved in numerous biological pathways. ACD’s RNA-protein workflow includes both a dual, or sequential ISH-IHC, detection method, as well as an integrated co-detection assay. View our recorded webinar on applying dual IHC/ISH methods below.