IHC immunohistochemistry protocol 

Introduction to IHC

Immunohistochemistry (IHC) is a widely used technique in research and diagnostics to detect specific antigens in tissue samples. This method combines histology and immunology, providing valuable insights into protein expression, tissue morphology, and disease mechanisms.

IHC Protocol: Step by Step

1. Tissue Preparation

• Fixation (formalin)
• Embedding (paraffin blocks)
• Sectioning (3–5 µm thin slides)

2. Deparaffinization and Rehydration

• Removal of paraffin with xylene
• Rehydration through graded ethanol series

3. Antigen Retrieval

• Heat-Induced Epitope Retrieval (HIER): citrate buffer pH 6.0 or Tris-EDTA pH 9.0
• Enzymatic Retrieval: proteinase K, trypsin

4. Blocking

• Block endogenous peroxidase activity
• Block non-specific binding with BSA or normal serum

5. Primary Antibody Incubation

• Apply specific primary antibody
• Incubate at room temperature or overnight at 4°C

6. Secondary Antibody and Detection

• Enzyme-linked (HRP/AP) or fluorescent secondary antibody
• Chromogenic detection (e.g., DAB) or fluorescence microscopy

7. Counterstaining and Mounting

• Hematoxylin counterstain for nuclei
• Dehydrate, clear, and mount with coverslip

8. Imaging and Analysis

• Light or fluorescence microscopy
• Digital image analysis for quantification

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Troubleshooting in IHC

Weak Staining

• Adjust antigen retrieval
• Increase antibody concentration

High Background

• Improve blocking steps
• Shorten antibody incubation

Non-Specific Staining

• Validate antibodies
• Use isotype controls

Applications of IHC

Cancer Research and Diagnostics

• Biomarkers like HER2, Ki-67, p53

Neuroscience

• Localization of neurotransmitters and receptors

Infectious Disease Studies

• Detection of viral and bacterial proteins

Drug Development

• Biomarker validation in clinical research

Conclusion

Immunohistochemistry is a key method in biomedical research and pathology, bridging molecular biology and clinical diagnostics. With a carefully optimized protocol, IHC delivers reproducible and meaningful results that support both scientific discovery and medical practice.

FAQ on IHC Protocol