Immunohistochemistry (IHC) is a vital technique in biomedical research and clinical diagnostics, enabling the visualization and localization of specific proteins within tissue samples. In this blog, we outline the different types of IHC staining, including direct and indirect approaches, immunofluorescence, and chromogenic techniques. We also discuss the general process of IHC staining. This overview serves as an introductory guide for understanding and implementing IHC protocols.

Types of IHC staining

Immunohistochemistry (IHC) staining is broadly categorized into several types based on the staining mechanism and the visualization methods used.

Direct Immunohistochemistry (Direct IHC): Involves the direct conjugation of the primary antibody with a detectable label (e.g., enzyme, fluorophore). This method is straightforward but may result in lower sensitivity compared to indirect methods.

Indirect Immunohistochemistry (Indirect IHC): Uses a secondary antibody that recognizes the primary antibody. The secondary antibody is conjugated to a detectable label, amplifying the signal from the primary antibody. This method enhances sensitivity and allows signal amplification.

Sandwich Immunohistochemistry: Involves using two primary antibodies targeting different epitopes on the same antigen. The first antibody binds to the antigen, and the second antibody binds to a different site on the antigen, sandwiching it. This method is useful for detecting antigens with multiple epitopes or when using antibodies that recognize denatured antigens.

Immunofluorescence (IF): Uses fluorophore-conjugated antibodies to visualize antigens under a fluorescence microscope. This technique is valuable for studying cellular localization, protein-protein interactions, and co-localization studies.

Chromogenic Immunohistochemistry: Utilizes enzyme-substrate reactions to produce a visible color change at the site of antibody-antigen binding. Commonly used enzymes include horseradish peroxidase (HRP) and alkaline phosphatase (AP). This method is widely used in clinical pathology and research.

These types of IHC staining methods provide flexibility in studying various aspects of protein expression and localization in tissues, catering to different research and diagnostic needs.

Steps of IHC staining

The general steps involved in immunohistochemistry (IHC) staining typically are described below. You can also browse available IHC reagents from Boster here.

  1. Tissue Preparation:
    • Fixation: Tissues are fixed using formaldehyde or other fixatives to preserve cellular structures and antigenicity.
    • Embedding: Tissues are embedded in paraffin or frozen in optimal cutting temperature (OCT) compound for sectioning.
  2. Antigen Retrieval:
    • Necessary for paraffin-embedded tissues to expose antigens masked during fixation. Techniques include heat-induced epitope retrieval (HIER) or enzymatic digestion.
  3. Blocking:
    • Non-specific binding sites on the tissue sections are blocked using a protein-based blocking buffer (e.g., serum from the same species as the secondary antibody).
  4. Primary Antibody Incubation:
    • Tissue sections are incubated with the primary antibody specific to the target antigen. Incubation time and conditions vary depending on the antibody and antigen characteristics.
  5. Washing:
    • Sections are washed to remove unbound primary antibodies and other reagents that could interfere with subsequent steps.
  6. Secondary Antibody Incubation:
    • Sections are incubated with a secondary antibody that recognizes the primary antibody. The secondary antibody is conjugated to a detectable label (e.g., enzyme or fluorophore).
  7. Amplification (if applicable):
    • Some protocols may include an amplification step, especially in indirect methods, to enhance signal detection.
  8. Washing:
    • Sections are washed again to remove unbound secondary antibodies and other reagents.
  9. Detection:
    • Depending on the staining method (e.g., chromogenic or fluorescent), a substrate or fluorophore is added to visualize the antigen-antibody complexes.
  10. Counterstaining (optional):
    • Counterstains like hematoxylin (for chromogenic) or DAPI (for fluorescent, catalog# AR1176) may be used to visualize cell morphology or nuclei.
  11. Mounting and Coverslipping:
    • Tissue sections are mounted on slides using a mounting medium to preserve the stained sections and coverslipped to protect them.
  12. Imaging and Analysis:
    • Stained tissue sections are examined under a microscope to analyze protein expression patterns, localization, and other relevant parameters.

These steps may vary slightly depending on the specific protocol, target antigen, and detection method used in the IHC staining process. Each step is crucial for obtaining reliable and reproducible results in immunohistochemistry studies. For a more comprehensive protocol, please visit our IHC Principle and IHC Protocol pages.

If you’d like to learn more about IHC, check out our IHC Technical Resource Center and download our IHC eBook, which discusses the IHC principle and protocol, and provides troubleshooting tips for your IHC experiment.

Mastering the intricacies of immunohistochemistry (IHC) staining techniques empowers researchers with powerful tools for elucidating protein expression and localization in tissues. Each step, from tissue preparation to antibody detection, plays a crucial role in ensuring accurate and reproducible results. By understanding the principles behind direct and indirect staining methods, as well as the nuances of immunofluorescence and chromogenic approaches, you will be able to tailor protocols to meet your specific research needs.