Essential Immunohistochemistry Techniques for Pathology Labs

Introduction

Immunohistochemistry (IHC) bridges histology and immunology by using antigen–antibody interactions to detect specific proteins within preserved tissue sections. Unlike routine hematoxylin & eosin stains, which highlight cell morphology, IHC reveals the presence or absence of diagnostic markers such as hormone receptors, oncoproteins and infectious agents. Pathologists rely on IHC to classify tumours, identify the tissue of origin and guide precision‑medicine therapies. Research laboratories use it to map protein distribution and study disease pathways. Because IHC preserves tissue architecture while visualising molecular targetspmc.ncbi.nlm.nih.gov, it provides a powerful window into tumour microenvironments and cellular interactions.

Despite its widespread use, IHC requires careful optimisation. Tissue fixation, sectioning and retrieval methods all influence antigenicity; antibodies vary in specificity; and detection systems differ in sensitivity. Errors in any step can produce false positives or negatives, undermining diagnosis. This in‑depth guide synthesises evidence‑based protocols from peer‑reviewed reviews, technical resourcesleicabiosystems.com and standards of practice. It outlines core techniques, highlights common pitfalls and explores emerging innovations such as multiplex IHC/immunofluorescence (mIHC/IF)pmc.ncbi.nlm.nih.gov. 

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Fundamentals of Immunohistochemistry

Principle of antigen–antibody staining

IHC visualises specific proteins in situ using primary antibodies raised against target antigens. These antibodies bind to epitopes within tissue sections. Secondary antibodies conjugated to enzymes (e.g., horseradish peroxidase or alkaline phosphatase) recognise the primary antibody and catalyse chromogenic reactions that produce coloured precipitates. Indirect methods using secondary antibodies amplify signal and therefore predominate in clinical practiceleicabiosystems.com.

Key advantages of IHC include high specificity and preservation of tissue architecture, allowing pathologists to assess both the intensity and localisation of staining relative to histological contextpmc.ncbi.nlm.nih.gov. However, antigen–antibody interactions can be masked by fixation, and non‑specific binding or endogenous enzymes may generate background staining. Thus, robust protocols and appropriate controls are critical.

Tissue handling, fixation and sectioning

Success begins when tissue is excised. Prolonged ischemia before fixation degrades proteins and activates enzymes, altering immunoreactivity. To prevent autolysis, surgical specimens should be immediately immersed in 10 % neutral buffered formalin (NBF) for around 24 hourspmc.ncbi.nlm.nih.gov. The ratio of tissue to fixative (typically 1 : 20) and the time should be consistent across cases. Frozen sections require cold acetone or NBF fixation for a few minutes when evaluating new antibodiespmc.ncbi.nlm.nih.gov.

After fixation, tissue is embedded in paraffin and sectioned. Standard thickness for IHC is 4 μmpmc.ncbi.nlm.nih.gov; thicker sections can yield higher background signal while thinner sections may lose antigen due to over‑exposure during deparaffinisation. Sections should be stored properly because extended storage (>2 months) can lead to antigen degradation such as loss of p53pmc.ncbi.nlm.nih.gov. Freshly cut sections are recommended for reproducible staining.

Step‑by‑Step Immunohistochemistry Protocol

The overall procedure of IHC follows a series of critical stepspmc.ncbi.nlm.nih.gov. Variations exist among laboratories, but the core workflow remains the same:

1. Deparaffinisation and hydration

Paraffin sections are deparaffinised in xylene and rehydrated through graded alcohols into water. Proper removal of paraffin ensures even reagent penetration. For frozen sections, this step is omitted.

2. Antigen (epitope) retrieval

Fixation with formalin cross‑links proteins and can mask epitopespmc.ncbi.nlm.nih.gov. Antigen retrieval breaks these cross‑links and restores antigenicity. Two main approaches exist:

• Heat‑induced epitope retrieval (HIER): The most widely used methodpmc.ncbi.nlm.nih.gov. Slides are placed in retrieval buffer (e.g., citrate pH 6.0, EDTA pH 8.0 or Tris–EDTA pH 9.0) and heated using a microwave, pressure cooker or water bath. Typical conditions range from 100 °C for 20–30 minutes or 120 °C for 10 minutespmc.ncbi.nlm.nih.gov. Over‑microwaving can destroy antigenicity and produce artefacts. Optimisation of buffer type, pH and time is essential for each antibody.

• Enzymatic retrieval: Some epitopes (e.g., certain cytokeratins) require proteolytic digestion. Sections are incubated with trypsin or proteinase at 37 °C for 10–20 minutespmc.ncbi.nlm.nih.gov. This method is harder to control and can damage tissue if not monitored. Empirical testing is necessary.

3. Blocking steps

To reduce background staining, several blocking steps are incorporated:

• Protein blocking: Non‑specific binding of the Fc portion of antibodies to tissue proteins can be prevented by incubating sections with 5–10 % serum from the same species as the secondary antibody or with protein buffers containing bovine serum albumin or gelatin. Commercial synthetic peptide mixes are also used. After blocking, thorough washing is necessary to remove excess protein.

• Endogenous enzyme blocking: When using horseradish peroxidase (HRP) detection, endogenous peroxidase activity must be quenched. Diluted hydrogen peroxide (3 %) is commonly appliedpmc.ncbi.nlm.nih.gov. For alkaline phosphatase systems, levamisole (10 mM) blocks endogenous alkaline phosphatase activity. Biotin‑rich tissues require avidin/biotin blocking to prevent interference. Adjust H₂O₂ concentration for sensitive antigens (e.g., 0.5 % for CD4)pmc.ncbi.nlm.nih.gov.

4. Primary antibody incubation

The primary antibody must be carefully selected and validated. A literature review helps identify proven clones. Monoclonal antibodies offer high specificity and reproducibility but may fail to recognise masked epitopes, whereas polyclonal antibodies are more sensitive yet can show higher background due to batch variabilitypmc.ncbi.nlm.nih.gov. Antibody validation requires positive and negative control tissue. Optimisation includes titration of antibody concentration, incubation time (usually 30–60 minutes at room temperature) and temperaturepmc.ncbi.nlm.nih.gov.

5. Secondary antibody and detection system

Most laboratories employ indirect detection. Secondary antibodies bind to the Fc region of primary antibodies; they are conjugated to enzymes that catalyse chromogenic reactions. Common systems include:

• Avidin–biotin complex (ABC) and labeled streptavidin–biotin (LSAB): Traditional methods but susceptible to endogenous biotin interference.

• Polymer‑based detection: Secondary antibodies conjugated to polymerised enzymes increase sensitivity by at least 50‑fold compared with standard methodspmc.ncbi.nlm.nih.gov. Polymer systems are preferred for low‑expressing antigens or small samples.

• Tyramide signal amplification (tyramine amplification): Utilises HRP to catalyse covalent deposition of fluorophore‑labeled tyramide, enabling multiple rounds of detection and facilitating multiplex IHC/IFpmc.ncbi.nlm.nih.gov.

The choice of detection system depends on tissue type and antigen expression. Horseradish peroxidase with diaminobenzidine (DAB) yields brown precipitate; alkaline phosphatase with Fast Red yields red product. AP is often preferred for tissues rich in endogenous peroxidase (e.g., bone marrow)pmc.ncbi.nlm.nih.gov.

6. Chromogen reaction and counterstaining

Substrates such as DAB or 3‑amino‑9‑ethylcarbazole (AEC) are added to visualise the enzyme activity. DAB produces an insoluble brown precipitate and is stable in organic solvents, making it suitable for permanent mounting. AEC yields red staining but is alcohol soluble; sections must be coverslipped with aqueous media.

After chromogen development, tissue is washed and counterstained. Hematoxylin is the most common nuclear counterstain; other options include eosin, methylene blue or methylene green depending on colour contrastpmc.ncbi.nlm.nih.gov. Counterstaining provides structural context and aids interpretation.

7. Mounting and analysis

Sections are dehydrated, cleared and mounted using appropriate media. Pathologists examine staining patterns—intensity, localisation (nuclear, cytoplasmic, membrane) and distribution—to make diagnoses. Digital slide scanners and image analysis software (e.g., FrediTech’s digital microscopy systems) facilitate quantification and remote consultations.

Antibody Validation and Quality Control

Importance of controls

IHC results are only meaningful when proper positive and negative controls are included. Standards from the Histochemical Society emphasise that an immunohistochemical assay lacking controls cannot be validly interpretedpmc.ncbi.nlm.nih.gov. Both false‑positive and false‑negative results can lead to erroneous diagnoses and wasted resources. Unfortunately, surveys of published literature revealed that up to 80 % of papers fail to mention controls and 89 % of journal author guidelines do not require thempmc.ncbi.nlm.nih.gov. To improve reproducibility, at least one positive and one negative control should be run with every batch.

Positive controls

Positive controls are specimens known to contain the target antigen in a specific location. Ideally, they are anatomical controls—tissues where the antigen is naturally expressed (e.g., pancreatic islets for insulin)pmc.ncbi.nlm.nih.gov. Positive cell lines or transfected cells are not recommended because they may lack dynamic range and calibration compared with tissuepmc.ncbi.nlm.nih.gov.

Negative controls

Negative controls show that staining results from specific antigen–antibody binding rather than non‑specific interactions. They include:

• Isotype control: Using an antibody of the same species and isotype as the primary antibody but lacking specificity for the antigen.

• No primary antibody control: Omitting the primary antibody to identify non‑specific binding by secondary antibodies or chromogenic reagents.

• Knockout tissue: When available, tissue from animals lacking the target antigen can demonstrate antibody specificity.

Antibody specificity and validation

Selecting validated antibodies reduces variability. Antibodies should be assessed by western blotting to confirm that they recognise a single protein band of expected sizepmc.ncbi.nlm.nih.gov. Even when manufacturers provide validation data, investigators must ensure specificity in their own tissues. Absorption controls—pre‑incubating antibody with antigenic peptide—are poor proxies for specificity and do not prove that staining identifies the intended targetpmc.ncbi.nlm.nih.gov.

Troubleshooting Common IHC Problems

Weak or absent staining

Possible causes include low antigen levels, over‑fixation, insufficient antigen retrieval or antibody concentration. Solutions include prolonging primary antibody incubation, optimising retrieval conditions and verifying the antibody’s shelf life.

Non‑specific or high background staining

Background may arise from endogenous enzyme activity, Fc receptor binding or non‑specific antibody binding. Ensure adequate blocking steps, dilute antibodies appropriately, extend washing times and use polymer‑based detection to reduce backgroundpmc.ncbi.nlm.nih.gov.

Edge staining or uneven staining

Uneven section adhesion or reagent application can produce gradients. Use charged slides, ensure complete section flattening and apply reagents uniformlyleicabiosystems.com. Avoid protein adhesives in flotation baths, as they can create pooling and inconsistent staining.

Over‑staining or under‑staining

Control chromogen development time; monitor DAB or AEC reaction under a microscope. Under‑staining may require longer incubation or higher antibody concentration; over‑staining can mask morphology.

Advanced Immunohistochemistry Techniques

Multiplex Immunohistochemistry/Immunofluorescence (mIHC/IF)

Traditional IHC labels one antigen per tissue section, limiting insight into complex microenvironments. mIHC/IF uses cyclic staining, tyramide signal amplification and spectral imaging to detect multiple markers on the same slide. A comprehensive review notes that multiplexed techniques circumvent the single‑marker limitation and allow simultaneous detection of multiple markers, enabling comprehensive study of cell composition, functional state and cell‑cell interactionspmc.ncbi.nlm.nih.gov. The technique offers high‑throughput staining and standardized quantitative analysis, providing reproducible and cost‑effective tissue studies. Meta‑analysis of 8135 patients found that mIHC/IF improved prediction of response to PD‑1/PD‑L1 immunotherapy compared to single‑marker tests like PD‑L1 IHC or tumour mutational burdenpmc.ncbi.nlm.nih.gov.

mIHC/IF platforms include tyramide‑based methods (e.g., Opal, Vectra), imaging mass cytometry (IMC) and multiplexed ion beam imaging (MIBI). These systems use different labels (fluorophores, metal tags) and detectors (spectral imaging, mass cytometry) to measure up to 40–100 markers simultaneously. Because they generate large datasets, automated image analysis and machine learning tools are essential. FrediTech’s digital microscopy solutions provide integration for scanning, aligning and analysing multiplex slides.

Immunofluorescence (IF)

IF is a variant of IHC in which secondary antibodies are conjugated to fluorophores instead of enzymes. IF enables simultaneous detection of multiple antigens by using spectrally distinct dyes and offers higher spatial resolution when combined with confocal or super‑resolution microscopy. However, fluorescence fades over time, and mounting requires anti‑fade reagents. IF is particularly useful in research to visualise protein co‑localisation.

Automated and digital immunohistochemistry

Automation improves reproducibility and throughput. Automated stainers standardise incubation times, retrieval conditions and washing, reducing human errorpmc.ncbi.nlm.nih.gov. However, manual staining allows flexibility when optimising new antibodies. Whole slide scanners coupled with image analysis software enable digital IHC quantification and remote consultations. FrediTech’s digital microscopy guide explains how digital cameras and image analysis algorithms integrate with microscopes for telepathology and AI‑assisted diagnosisfreditech.com.

Multiplex immunohistochemistry in cancer immunotherapy

In the era of immune checkpoint inhibitors, assessing multiple immune and tumour markers within the same section is crucial. mIHC/IF allows pathologists to evaluate spatial relationships between tumour cells, cytotoxic T lymphocytes, regulatory T cells and checkpoint ligands. For example, by staining for PD‑L1, PD‑1, CD3, CD8 and cytokeratin, pathologists can compute tumour proportion scores and immune infiltration patternspmc.ncbi.nlm.nih.gov. The ability to preserve scarce tissues while extracting complex immunoprofiles enhances diagnostic value and informs therapeutic decisions.

Integration with other technologies

Newer methods such as imaging mass cytometry label antibodies with metal isotopes and detect them using time‑of‑flight mass spectrometry, enabling simultaneous quantification of >40 markers without spectral overlap. Digital spatial profiling combines oligonucleotide‑tagged antibodies with next‑generation sequencing to measure up to 60 markers and generate spatially resolved gene‑expression datapmc.ncbi.nlm.nih.gov. As these platforms mature, pathologists will need to coordinate with molecular scientists to interpret multi‑modal data. FrediTech’s advanced imaging page highlights how breakthroughs in sensors and computing power enable sophisticated imaging modalitiesfreditech.com.

Step‑by‑Step Guide for Pathology Labs

Implementing a robust IHC workflow requires planning and quality assurance. The following steps summarise best practices:

1. Specimen collection and fixation: Immediately fix tissue in 10 % NBF for 24 hours; ensure proper tissue-to-fixative ratiopmc.ncbi.nlm.nih.gov.
2. Embedding and sectioning: Embed in paraffin, cut 4 μm sections and mount onto charged slides to promote adhesion. Store slides in a desiccator and avoid storage beyond two monthspmc.ncbi.nlm.nih.gov.
3. Deparaffinisation: Use xylene and graded ethanol to remove paraffin and rehydrate sections.
4. Antigen retrieval: Select HIER or enzymatic retrieval based on antigen; optimise buffer pH, temperature and time.
5. Blocking: Perform protein block with appropriate serum or protein buffer; quench endogenous peroxidase and phosphatase; block biotin when necessarypmc.ncbi.nlm.nih.gov.
6. Primary antibody incubation: Choose validated monoclonal or polyclonal antibodies; titrate concentration and incubation time; include appropriate positive and negative control slides.
7. Secondary antibody and detection: Apply the chosen detection system (polymer‑HRP, polymer‑AP, ABC, etc.) and incubate as recommendedpmc.ncbi.nlm.nih.gov.
8. Chromogen development: Add DAB or AEC until the desired intensity is achieved; monitor under a microscope to avoid overstaining. Stop the reaction with distilled water.
9. Counterstaining: Immerse slides in hematoxylin for about one minute; differentiate if necessary; rinse and blue in running water.
10. Dehydration and mounting: Dehydrate through graded ethanol and xylene; coverslip using appropriate mountant.
11. Evaluation and documentation: Assess staining pattern and intensity. Use digital imaging and AI‑based quantification when available. Document all parameters (antibody clone, lot number, dilution, retrieval method) for reproducibility.

Real‑World Examples and Applications

Breast cancer receptor testing

IHC is integral to determining oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) status in breast cancer. These biomarkers guide endocrine therapy and HER2‑targeted treatments. Although FISH is considered a reference test for HER2 gene amplification, IHC remains a convenient screening method with high sensitivity and specificity when optimisedpmc.ncbi.nlm.nih.gov. The combination of IHC and in situ hybridisation ensures accurate classification.

PD‑L1 testing for immunotherapy

PD‑L1 IHC assays determine eligibility for anti‑PD‑1/PD‑L1 therapies. Multiplex IHC/IF can simultaneously evaluate PD‑L1 expression with immune markers like CD3 and CD8 to predict response. Meta‑analysis has shown that mIHC/IF better predicts treatment outcomes than PD‑L1 IHC alonepmc.ncbi.nlm.nih.gov.

Infectious disease diagnostics

IHC aids detection of viral, bacterial and fungal antigens in tissue. For example, cytomegalovirus inclusions in transplant biopsies or mycobacterial antigens in granulomas can be visualised, guiding treatment.

Research and biomarker discovery

In research, IHC localises novel biomarkers, assesses drug effects and validates gene‑expression data. High‑throughput multiplex IHC/IF platforms enable spatial profiling of tumour microenvironments and help discover predictors of therapy response. Combined with FrediTech’s digital microscopy and advanced imaging solutionsfreditech.com freditech.com, researchers can share images, integrate AI‑based quantification and collaborate remotely.

Innovations and Future Directions

Digital pathology and AI

Whole slide imaging scanners convert slides into high‑resolution digital images. Combined with AI algorithms, digital pathology can automate quantification of staining intensity, reduce inter‑observer variability and highlight regions of interest. FrediTech’s guide to digital microscopy explains how digital cameras and software enable sharing, annotation and analysis of imagesfreditech.com.

Machine learning models trained on annotated slides can score markers such as ER, PR and Ki‑67, providing reproducible results. Deep learning–based image analysis is being integrated into IHC workflows for tumour grading and prognostic prediction.

3D and whole‑organ IHC

Tissue clearing techniques (e.g., CLARITY, iDISCO) combined with light‑sheet microscopy allow immunostaining of intact tissues. By rendering tissues transparent, these methods enable 3D visualisation of immune cell infiltration and tumour architecture. Multiplex staining and antibody penetration in cleared tissues remain challenging but hold promise for future pathology.

Automated multiplex platforms

Commercial systems like Leica Bond RX and Akoya Vectra automate cyclic staining and imaging for mIHC/IF. These platforms support up to seven or more markers with spectral unmixing. Imaging mass cytometry (Fluidigm Hyperion) and multiplexed ion beam imaging (IonPath MIBI) expand marker panels to over 40, though they require specialised instrumentation and computational analysis.

Integration with omics

Digital spatial profiling and other spatial transcriptomics technologies complement IHC by measuring mRNA or protein expression in situ. Future pathology labs will integrate proteomic, transcriptomic and genomic data to provide holistic diagnoses.

Conclusion

Immunohistochemistry remains a cornerstone of diagnostic pathology and biomedical research because it detects specific proteins while preserving tissue architecture. However, accurate and reproducible results require meticulous attention to pre‑analytic variables, optimisation of antigen retrieval, careful antibody selection, appropriate detection systems and rigorous quality control. The step‑by‑step protocols and troubleshooting tips provided here draw from validated guidelinespmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov and emphasise the importance of positive and negative controlspmc.ncbi.nlm.nih.gov. Pathology laboratories that implement these best practices improve diagnostic confidence and patient care.

Innovations such as multiplex IHC/IF and digital pathology are transforming the field by enabling simultaneous detection of multiple markers, high‑throughput analysis and integration with AI. As these technologies mature, pathologists will need to adapt workflows and collaborate with informatics experts. 

By combining rigorous methodology with emerging technologies, pathology labs can harness immunohistochemistry to uncover disease mechanisms, stratify patients for targeted therapies and contribute to scientific discovery. Continuous education, adherence to quality guidelines and collaboration with industry partners will ensure that IHC remains a powerful tool for decades to come.

Frequently Asked Questions (FAQ)

What is immunohistochemistry and why is it important?

Immunohistochemistry (IHC) is a technique that uses antibodies to visualise specific proteins within tissue sections. It helps pathologists classify tumours, confirm diagnoses and guide targeted treatments. Unlike routine stains, IHC provides molecular information while retaining tissue architecturepmc.ncbi.nlm.nih.gov.

What are the main steps in an IHC protocol?

Key steps include tissue fixation, embedding and sectioning, deparaffinisation, antigen retrieval, blocking of non‑specific binding and endogenous enzymes, incubation with primary and secondary antibodies, chromogen development, counterstaining and mountingpmc.ncbi.nlm.nih.gov. Each step must be optimised for reproducible results.

How do I choose between monoclonal and polyclonal antibodies?

Monoclonal antibodies recognise a single epitope and offer high specificity and reproducibility, but may be less sensitive to masked epitopes. Polyclonal antibodies recognise multiple epitopes and are more sensitive but can produce higher background due to batch variabilitypmc.ncbi.nlm.nih.gov.

Why is antigen retrieval necessary?

Formalin fixation masks epitopes by cross‑linking proteins. Antigen retrieval reverses this masking, restoring epitope accessibility and improving antibody bindingpmc.ncbi.nlm.nih.gov. Heat‑induced epitope retrieval (HIER) is most common, using buffers heated in microwaves or pressure cookerspmc.ncbi.nlm.nih.gov.

What controls are essential in IHC?

At least one positive control (tissue known to express the antigen) and one negative control (isotype or no primary antibody) must accompany each run. Controls ensure that staining results from specific antigen–antibody interactions and help identify technical issuespmc.ncbi.nlm.nih.gov.

What is multiplex immunohistochemistry/immunofluorescence?

mIHC/IF allows simultaneous detection of multiple biomarkers in a single tissue section using cyclic staining and advanced detection systems. It enables comprehensive profiling of tumour microenvironments and improves prediction of immunotherapy responsepmc.ncbi.nlm.nih.govpmc.ncbi.nlm.nih.gov.

How is digital pathology changing IHC?

Digital slide scanners convert glass slides into high‑resolution images that can be analysed, shared and archived. AI algorithms can quantify staining and identify patterns, reducing observer variability. FrediTech’s digital microscopy resources provide guidance on implementing these workflowsfreditech.com.

Can IHC be automated?

Yes. Automated stainers standardise retrieval, blocking, antibody incubation and washing steps, improving reproducibilitypmc.ncbi.nlm.nih.gov. Automation is ideal for high‑throughput diagnostic labs. Manual staining remains useful for research and novel antibodies requiring optimisation.

How do I troubleshoot weak or non-specific staining?

Check antigen retrieval conditions, antibody concentration and incubation times. Ensure appropriate blocking steps and consider using polymer‑based detection to enhance sensitivitypmc.ncbi.nlm.nih.gov. Evaluate slide quality and section adhesionleicabiosystems.com.

Where can I learn more about digital microscopy and advanced imaging?

Visit FrediTech’s blog posts on digital microscopy, advanced imaging techniques and equipment selection for comprehensive insights into imaging technologies and how they integrate with IHC workflows. Their resources help laboratories choose the right microscopes, cameras and software to achieve optimal results.freditech.comfreditech.com

Author Credentials

Wiredu Fred is a medical laboratory scientist and health technology writer. With over a decade of experience in clinical pathology, digital imaging and laboratory management, Fred combines practical knowledge with a passion for science communication. He regularly contributes to FrediTech’s blog, offering insights into laboratory equipment, microscopy and diagnostic innovations.