Overview
Immunohistochemistry (IHC) is a powerful diagnostic technique that uses antibodies to detect specific proteins in tissue sections. This technique has revolutionized diagnostic pathology by allowing pathologists to identify cell types, determine tumor origin, assess prognosis, and predict treatment response. IHC combines the specificity of antibodies with the morphological context of tissue sections, providing both diagnostic and prognostic information.
IHC is essential for modern diagnostic pathology, particularly in oncology where it helps classify tumors, determine primary site of metastatic disease, and guide targeted therapy. It is crucial for distinguishing between different types of lymphoma, identifying hormone receptors in breast cancer, detecting microsatellite instability in colorectal cancer, and assessing programmed death-ligand 1 (PD-L1) expression for immunotherapy. The technique also plays a vital role in research, drug development, and understanding disease mechanisms.
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Indications
Identification of tumor type and origin using lineage-specific markers
Determination of primary site in cases of metastatic cancer of unknown origin
Classification of lymphoid neoplasms using B-cell, T-cell, and myeloid markers
Evaluation of estrogen and progesterone receptors in breast cancer
Assessment of HER2, PD-L1, and other therapeutic targets
Evaluation of Ki-67, p53, and other prognostic indicators
Contraindications
Absolute Contraindications
Relative Contraindications
📋 Equipment Checklist
Check off items as you gather them:
Pre-procedure Preparation
Preparation involves ensuring the tissue sections are properly fixed and embedded in paraffin. Sections should be cut at 3-4 microns and mounted on charged slides to prevent detachment during processing. The automated staining platform should be calibrated and reagents should be at room temperature. Primary antibodies should be validated and stored according to manufacturer recommendations. Antigen retrieval solutions should be prepared fresh or used within their expiration date.Step-by-Step Procedure
Step 1: Slide Preparation and Deparaffinization
Cut tissue sections at 3-4 microns and mount on charged slides. Allow slides to dry overnight at 37°C or for 1 hour at 60°C. Deparaffinize slides by immersing in xylene for 5 minutes, followed by two changes of 100% alcohol for 3 minutes each. Rehydrate through graded alcohols (95%, 70%, 50%) and rinse in distilled water. Ensure complete removal of paraffin to prevent background staining.
⚠️ Common Mistakes to Avoid:
- Incomplete deparaffinization causing background staining
- Inadequate drying time causing section detachment
- Using tap water instead of distilled water
💡 Pro Tip:
Check for complete deparaffinization by ensuring the tissue appears translucent and not opaque.
Step 2: Antigen Retrieval
Perform antigen retrieval to unmask epitopes that may have been masked during formalin fixation. For heat-induced epitope retrieval (HIER), immerse slides in preheated citrate buffer (pH 6.0) or EDTA (pH 8.0) and heat in a pressure cooker or microwave for 20-30 minutes. Allow slides to cool for 20 minutes. For enzyme-induced epitope retrieval, use proteinase K or trypsin for 5-15 minutes at 37°C.
⚠️ Common Mistakes to Avoid:
- Incorrect pH of retrieval buffer for specific antibodies
- Overheating causing tissue damage
- Insufficient cooling time before proceeding
💡 Pro Tip:
Different antibodies require different antigen retrieval methods - always check the antibody datasheet for specific requirements.
Step 3: Endogenous Peroxidase Blocking
Block endogenous peroxidase activity by immersing slides in 3% hydrogen peroxide in methanol for 10 minutes. This prevents false positive staining from endogenous peroxidase in red blood cells and other tissues. Rinse slides thoroughly in distilled water and then in phosphate-buffered saline (PBS).
⚠️ Common Mistakes to Avoid:
- Inadequate blocking causing background staining
- Using hydrogen peroxide that is too old or diluted
- Insufficient rinsing after blocking
💡 Pro Tip:
For alkaline phosphatase-based detection systems, use levamisole or other appropriate blocking agents instead of hydrogen peroxide.
Step 4: Protein Blocking
Block non-specific protein binding sites by applying normal serum from the same species as the secondary antibody. Apply 100-200 μL of 5-10% normal serum in PBS for 10-15 minutes. This prevents non-specific binding of the primary and secondary antibodies to tissue proteins.
⚠️ Common Mistakes to Avoid:
- Using serum from the wrong species
- Inadequate blocking time
- Using serum that is too old or contaminated
💡 Pro Tip:
For some antibodies, commercial blocking solutions may be more effective than normal serum.
Step 5: Primary Antibody Application
Apply the primary antibody at the appropriate dilution in PBS or antibody diluent. Use 100-200 μL per slide and incubate at room temperature for 30-60 minutes or overnight at 4°C. Ensure the antibody covers the entire tissue section. For automated stainers, follow the manufacturer's protocol for antibody application.
⚠️ Common Mistakes to Avoid:
- Using incorrect antibody dilution
- Insufficient incubation time
- Allowing slides to dry during incubation
💡 Pro Tip:
Always include positive and negative controls with each staining run to validate the results.
Step 6: Secondary Antibody and Detection
Rinse slides thoroughly in PBS to remove unbound primary antibody. Apply the secondary antibody conjugated to horseradish peroxidase (HRP) or alkaline phosphatase (AP) for 30 minutes at room temperature. Rinse again and apply the chromogen (DAB for HRP, Fast Red for AP) for 5-10 minutes. Monitor the reaction under the microscope to avoid overstaining.
⚠️ Common Mistakes to Avoid:
- Inadequate rinsing between steps
- Overstaining with chromogen
- Using expired secondary antibody
💡 Pro Tip:
For automated systems, the detection steps are usually performed automatically according to the manufacturer's protocol.
Step 7: Counterstaining and Mounting
Rinse slides thoroughly in distilled water to stop the chromogen reaction. Apply hematoxylin counterstain for 30-60 seconds, rinse in water, and differentiate in acid alcohol if needed. Dehydrate through graded alcohols, clear in xylene, and mount with coverslip using appropriate mounting medium.
⚠️ Common Mistakes to Avoid:
- Over-counterstaining obscuring positive staining
- Inadequate dehydration causing poor slide quality
- Using inappropriate mounting medium
💡 Pro Tip:
For fluorescent IHC, use appropriate mounting medium that preserves fluorescence and prevents photobleaching.
Step 8: Quality Control and Interpretation
Examine stained slides under the microscope to assess staining quality and specificity. Check that positive controls show appropriate staining and negative controls show no staining. Evaluate the staining pattern, intensity, and distribution. Document results and correlate with morphological findings and clinical information.
⚠️ Common Mistakes to Avoid:
- Not checking controls before interpreting results
- Ignoring background staining
- Not correlating with morphological findings
💡 Pro Tip:
Always interpret IHC results in the context of the clinical history and other diagnostic studies.
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Post-procedure Care
Post-staining care involves proper documentation of staining results, quality control assessment, and correlation with morphological findings. Stained slides should be coverslipped and stored appropriately. Results should be interpreted in the context of clinical information and other diagnostic studies. Quality assurance measures include regular validation of antibodies, monitoring of positive and negative controls, and participation in external quality assessment programs.Complications & Management
| Complication | Incidence | Signs | Management | Prevention |
|---|---|---|---|---|
| Background staining | 5-15% | Non-specific staining throughout the tissue | Optimize blocking, adjust antibody dilution, improve antigen retrieval | Proper blocking, appropriate antibody dilution, good technique |
| False negative results | 2-8% | Expected positive staining is absent | Check antigen retrieval, antibody validation, tissue quality | Proper antigen retrieval, validated antibodies, good tissue fixation |
| False positive results | 1-5% | Unexpected positive staining | Check antibody specificity, use appropriate controls | Validated antibodies, proper controls, good technique |
| Section detachment | 3-10% | Tissue sections fall off slides during processing | Recut sections, use charged slides, improve technique | Charged slides, proper drying, gentle handling |
| Weak staining | 5-12% | Faint or barely visible positive staining | Optimize antigen retrieval, increase antibody concentration | Proper antigen retrieval, appropriate antibody dilution |
Clinical Pearls
Always include positive and negative controls with each staining run to validate the results and detect technical problems.
The choice of antigen retrieval method (citrate vs EDTA) can significantly affect staining results - citrate is better for most antibodies, while EDTA is preferred for some nuclear antigens.
For automated staining platforms, always follow the manufacturer's protocol exactly - deviations can lead to inconsistent results.
Always interpret IHC results in the context of morphological findings - IHC should support, not replace, morphological diagnosis.
Document the antibody clone, dilution, and incubation conditions for each staining run for quality assurance and troubleshooting.
For panels of antibodies, stain them in the same run to ensure consistent conditions and allow direct comparison of staining patterns.
Be aware of cross-reactivity between antibodies - some antibodies may react with unexpected antigens, leading to false positive results.