Overview
Immunohistochemistry (IHC) identifies specific cellular or tissue antigens using labelled antibodies, allowing pathologists to detect proteins that morphology alone cannot reveal. It has become an essential tool for tumour classification, prognostication, and treatment selection.
This lesson explains antigen-antibody binding concepts such as affinity, avidity, and specificity, describes polyclonal versus monoclonal antibodies, outlines direct and indirect detection methods, and covers antigen retrieval techniques used to unmask antigens hidden by formalin fixation.
Learning Objectives
After this lesson you will be able to…- Explain the principle of antigen-antibody interaction in immunohistochemistry
- Differentiate affinity, avidity, specificity, and sensitivity
- Compare polyclonal and monoclonal antibodies as primary reagents
- Describe direct, indirect, and polymer-based IHC detection methods
- Explain the purpose and methods of antigen retrieval
Clinical Story
Why This MattersA breast biopsy shows an infiltrating carcinoma on H&E, but the oncologist needs to know the tumour's hormone receptor and HER2 status before deciding on treatment. The laboratory performs immunohistochemistry for oestrogen receptor, progesterone receptor, and HER2, results that will directly determine whether the patient receives hormonal therapy, targeted therapy, or chemotherapy.
Core Concepts
Affinity describes the three-dimensional fit and binding strength between an antibody and its antigen, while avidity refers to the combined functional strength when an antibody binds multiple epitopes. Antibody specificity is the ability to bind selectively to a single epitope, and sensitivity is the smallest amount of antigen a technique can detect.
Polyclonal antibodies are harvested from an immunised animal's serum and represent a mixture of antibodies against multiple epitopes, sometimes causing cross-reactivity. Monoclonal antibodies are produced by the hybridoma method, fusing an antibody-producing plasma cell with an immortal myeloma cell line, yielding a highly specific, unlimited supply of a single antibody clone.
IHC methods range from the simple direct technique (label conjugated directly to the primary antibody) to more sensitive indirect and polymer-based methods (e.g. EnVision) that amplify the signal. Antigen retrieval, performed by heat (microwave, pressure cooker, steamer, water bath) or enzymatic digestion, reverses formalin-induced protein cross-linking to expose hidden antigenic sites.
Laboratory Principle
IHC relies on the lock-and-key specificity of antibody variable domains for their target antigen. A labelled antibody (directly or via a secondary antibody/polymer) binds the target antigen in the tissue section, and an enzyme label such as horseradish peroxidase converts a chromogen substrate (commonly DAB) into a stable, visible coloured precipitate at the site of binding, allowing light microscopic localisation of the antigen.
Equipment Required
Reagents & Materials
| Reagent / Material | Concentration / Grade | Purpose | Storage |
|---|---|---|---|
| Sodium citrate buffer (pH 6.0) | 10mM tri-sodium citrate + 0.05% Tween 20 | Heat-induced antigen retrieval | Room temperature, 3 months |
| Primary antibody | Manufacturer-recommended dilution in TBS/1% BSA | Binds specific tissue antigen | 4°C, per datasheet |
| DAB chromogen (3,3'-diaminobenzidine) | Working strength per kit instructions | Produces brown reaction end-product with HRP | Prepare fresh, light-protected |
Step-by-Step Procedure
Bring sections to water, then perform heat-induced or enzymatic antigen retrieval to unmask antigens hidden by formalin fixation.
Incubate in hydrogen peroxide to quench endogenous peroxidase, then block with normal serum/BSA for 1-2 hours.
Apply the primary antibody at the appropriate dilution and incubate overnight at 4°C in a humidified chamber.
Rinse, then apply an enzyme-labelled secondary antibody or polymer detection system and incubate for about 1 hour at room temperature.
Develop with chromogen (e.g. DAB) for around 10 minutes, counterstain if required, then dehydrate, clear, and mount with DPX.
Flow Diagram
Quality Control
Include a known positive control tissue expressing the target antigen and a negative control (primary antibody omitted) with every IHC run to confirm specific staining and rule out non-specific background.
Participate in external IHC quality assurance schemes (e.g. for hormone receptor or HER2 testing) to verify inter-laboratory reproducibility, particularly for antibodies guiding treatment decisions.
Reference Values
Normal Ranges⚠️ Reference ranges may vary between laboratories. Always apply your laboratory's established reference intervals.
Clinical Interpretation
| Finding | Possible Significance | Action / Follow-up |
|---|---|---|
| Strong, specific brown membrane/nuclear/cytoplasmic staining | Positive for target antigen (e.g. hormone receptor, HER2) | Report according to standardised scoring criteria for the specific marker |
| Diffuse background staining without true positive control confirmation | Non-specific binding or inadequate blocking | Repeat with proper blocking and negative control to confirm result validity |
| No staining in known positive control | Technical failure — antibody, retrieval, or detection system issue | Investigate reagent expiry, retrieval method and repeat the entire run |
Common Errors & How to Avoid Them
Cause: Inadequate blocking of non-specific binding sites, or drying of sections during incubation.
Prevention: Ensure adequate blocking with serum/BSA and keep all incubations in a humidified chamber to prevent drying.
Cause: Inadequate or absent antigen retrieval, allowing formalin cross-links to mask the antigen.
Prevention: Optimise antigen retrieval method and duration for each antibody, and always run a known positive control.
Cause: Primary antibody used at incorrect dilution or past its expiry.
Prevention: Titrate primary antibody dilution against the manufacturer's datasheet and use fresh reagents within their validity period.
Laboratory Tips from the Bench
Never skip the humidified chamber step — drying at any stage causes non-specific binding and unacceptably high background.
Always run a positive and a negative control with every IHC batch; a stain without controls cannot be reliably interpreted.
Remember: 'Retrieve before you receive' — antigen retrieval must always precede antibody application, since it reverses the very cross-linking that formalin fixation created.
Important Notes
Heat-based antigen retrieval methods (microwave, pressure cooker, steamer, water bath) have significantly improved immunohistochemical sensitivity compared to older enzymatic-only methods.
Newer polymer-based detection systems (e.g. EnVision) attach multiple antibodies and enzyme molecules to a dextran backbone, greatly amplifying the signal compared to the traditional direct method.
Interactive Quiz
Test Your KnowledgeFlashcards
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Clinical Case Study
Apply Your KnowledgePresents with a palpable breast lump. Core biopsy confirms infiltrating ductal carcinoma on H&E; oncologist requests hormone receptor and HER2 testing before treatment planning.
Strong ER and PR positivity with a negative HER2 result indicates a hormone receptor-positive, HER2-negative breast carcinoma, making the tumour a candidate for hormonal (endocrine) therapy rather than HER2-targeted therapy.
- →IHC results directly guide systemic treatment selection in breast cancer.
- →Antigen retrieval and proper controls are essential for reliable hormone receptor testing.
- →HER2 status determines eligibility for targeted anti-HER2 therapy.
Frequently Asked Questions
It confirms that any staining seen is due to specific primary antibody binding and not non-specific binding of the secondary antibody or detection system.
In the direct technique the label is conjugated to the primary antibody itself, while the indirect technique uses a separate labelled secondary antibody against the primary, providing greater signal amplification and sensitivity.
A pressure cooker heats more uniformly than a microwave, avoiding hot and cold spots, and the higher temperature achievable under pressure (around 120°C) can improve antigen unmasking for some antibodies.
Quick Revision
10-Minute ReviewKey Takeaways
- Immunohistochemistry visualises specific antigens using antibody-based detection.
- Affinity, avidity, specificity, and sensitivity describe distinct aspects of antibody performance.
- Monoclonal antibodies offer superior specificity compared to polyclonal antibodies.
- Polymer-based detection systems amplify signal beyond traditional direct methods.
- Antigen retrieval is essential for formalin-fixed, paraffin-embedded tissue.
- IHC results, such as hormone receptor and HER2 status, directly influence patient treatment.
Competency Checklist
Track Your MasteryReferences
- NIOS Histology and Cytology Practical Manual, Lesson 18: Immunohistochemistry.
- Dabbs DJ. Diagnostic Immunohistochemistry. 5th ed.
- Bancroft JD, Gamble M. Theory and Practice of Histological Techniques. 7th ed.