Immunohistochemistry

Histology: Tissue Prep - Slice of Life

• Fixation: Preserves tissue structure, typically with formalin.
• Dehydration: An ascending series of alcohol removes water.
• Clearing: Xylene makes the tissue transparent by removing alcohol.
• Infiltration: The tissue is permeated with molten paraffin wax.
• Embedding: The sample is placed in a wax block for support.
• Sectioning: A microtome cuts the block into 4-5 µm thick sections.

⭐ Formalin creates covalent cross-links between proteins (lysine residues), which preserves tissue structure but can also mask antigenic sites, requiring antigen retrieval for IHC.

Basic Staining - The Color Purple

• Hematoxylin (H): A basic dye that stains acidic, basophilic structures blue.
  • Stains: Nucleus (DNA/RNA), rough endoplasmic reticulum (rER), ribosomes.
• Eosin (E): An acidic dye that stains basic, eosinophilic (acidophilic) structures pink/red.
  • Stains: Cytoplasm, mitochondria, collagen, muscle.

📌 Basophilic = Blue (attracts basic dyes). Acidophilic = Pink/Red (attracts acidic dyes like eosin).

⭐ Nissl bodies (rER in neurons) are basophilic, staining dark blue with H&E.

IHC Principles - Tag, You're It!

• Goal: To visualize a specific protein (antigen) within a tissue slice using an antibody-based system.
• Core Components:
  • Antigen (Ag): The target protein.
  • Primary Antibody (1° Ab): Binds to the antigen.
  • Secondary Antibody (2° Ab): Binds the 1° Ab, carrying an enzyme.
  • Enzyme: Horseradish Peroxidase (HRP) is the workhorse.
  • Chromogen: Diaminobenzidine (DAB) is catalyzed by HRP to produce a stable, visible brown precipitate.

⭐ The indirect IHC method is more sensitive than the direct method because multiple secondary antibodies can bind to a single primary antibody, amplifying the signal.

Clinical IHC Markers - Cancer's Calling Card

Immunohistochemistry (IHC) uses antibodies to detect specific antigens (proteins) in tissue samples, acting as a "calling card" for different cell types. This is crucial for diagnosing and classifying neoplasms, especially when morphology on H&E stain is ambiguous. It also guides targeted therapy and helps establish a prognosis. The choice of markers is tailored to the differential diagnosis based on the tumor's location and appearance.

High‑Yield Points - ⚡ Biggest Takeaways

• Immunohistochemistry (IHC) uses antigen-antibody binding to visualize specific proteins in tissue, aiding in tumor diagnosis.
• Cytokeratin is the classic marker for tumors of epithelial origin (carcinomas).
• Vimentin is the characteristic marker for tumors of mesenchymal origin (sarcomas).
• S-100 is a key marker for cells of neural crest origin, such as melanoma and Schwannoma.
• CD markers (e.g., CD20, CD3) are essential for classifying lymphomas and leukemias.
• GFAP is a specific marker for glial cells and their tumors (e.g., astrocytoma).