免疫組織化学と免疫細胞化学:光顕微鏡による組織イメージング
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Q1: What is the main difference between immunohistochemistry and immunocytochemistry?
Immunohistochemistry (IHC) visualizes proteins in thin slices of whole tissue sections, while immunocytochemistry (ICC) visualizes proteins in isolated or cultured cells. Both techniques use antibodies tagged with detection systems to identify and localize target proteins. The key difference lies in the sample type: IHC maintains tissue structure, whereas ICC shows antigen distribution within individual cells without tissue context.
Q2: Why is sample preparation different between immunohistochemistry and immunocytochemistry?
ICC requires three preparation steps: plating cells on coverslips, fixation with paraformaldehyde, and permeabilization with detergent. IHC requires five steps: fixation, paraffin embedding, sectioning with a microtome, deparaffinization, and optional antigen retrieval. These differences exist because IHC must preserve tissue structure through embedding and sectioning, while ICC only needs to prepare individual cells for antibody access.
Q3: What role does the secondary antibody play in immunohistochemistry staining?
The secondary antibody binds to the primary antibody that has already attached to the target protein. It is conjugated to HRP enzyme, which converts the DAB substrate into an insoluble brown precipitate. This brown stain marks the exact location of the target protein, allowing visualization under light microscopy. The secondary antibody essentially amplifies the signal from the primary antibody.
Q4: How does antigen retrieval improve immunohistochemistry results?
Antigen retrieval uses heat or enzymes to unmask epitopes that were cross-linked during fixation with paraformaldehyde. This unmasking makes epitopes available for antibody binding, improving staining specificity and intensity. Without antigen retrieval, cross-linked proteins may remain inaccessible to antibodies, resulting in weak or false-negative staining in paraffin-embedded tissue sections.
Q5: What is the purpose of hematoxylin counterstaining in immunohistochemistry?
Hematoxylin counterstaining labels cell nuclei blue, providing a spatial reference point for determining subcellular localization of the target protein. The blue nuclear stain contrasts with the brown DAB precipitate marking the target protein, allowing researchers to assess whether protein expression is nuclear, cytoplasmic, or membrane-localized. This dual staining improves anatomical context and interpretation.
Q6: How does deparaffinization prepare tissue sections for immunohistochemistry staining?
Deparaffinization removes the paraffin wax surrounding tissue sections using xylene, followed by rehydration through graded ethanol solutions. This process restores tissue hydration and allows antibodies to penetrate the tissue. Without deparaffinization, antibodies cannot access antigens within the paraffin-embedded tissue, making staining impossible.
Q7: Why is blocking buffer used before adding primary antibodies in immunohistochemistry?
Blocking buffer, typically horse serum diluted in PBS, prevents non-specific antibody binding to tissue components. It saturates potential binding sites on the tissue, ensuring that only the primary antibody specific to the target protein binds. This step reduces background staining and improves signal-to-noise ratio, making the brown DAB precipitate marking the target protein more visible and interpretable.