Sample interview questions: Can you explain the principles of tissue immunohistochemistry staining and interpretation?
Sample answer:
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Principles of Tissue Immunohistochemistry Staining:
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Antigen Retrieval: This step aims to expose hidden or masked antigens in the tissue sections by breaking cross-links or unmasking epitopes.
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Blocking: Blocking agents like bovine serum albumin (BSA) or normal serum are used to reduce background staining and minimize non-specific antibody binding.
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Primary Antibody Incubation: Specific primary antibodies, raised against the target protein of interest, are applied to the tissue sections. The primary antibodies bind to their specific antigens.
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Secondary Antibody Incubation: Labeled secondary antibodies, which recognize the primary antibody species, are applied. These secondary antibodies are conjugated with enzymes (e.g., horseradish peroxidase) or fluorescent molecules.
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Detection: The enzymatic reaction or fluorescent signal generated by the labeled secondary antibodies allows visualization of the target protein’s location and distribution within the tissue section.
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Interpretation of Immunohistochemistry Staining:
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Positive Staining: The presence of a colored reaction product (in case of enzymatic labeling) or fluorescent signal indicates the presence of the target protein.
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Negative Staining: The absence of any staining or a very weak signal suggests the absence or low levels of the target protein.
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Specificity: Careful selection of antibodies and appropriate controls are crucial to ensure the specificity of the staining. Positive and negative controls help eliminate false-positive or false-negative results.
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