What should I consider when choosing an immunohistochemistry (IHC) antibody?
Posted May 2, 2024
Answer
Consider these factors when choosing a primary antibody for immunohistochemistry (IHC) experiments:
- Animal species in which the primary antibody is raised: The species of animal in which the primary antibody is raised is important in multiplexing experiments to prevent cross-reactivity with the corresponding secondary antibodies.
- Antibody labeling: Before you start staining, determine if the primary antibody you are using is directly labeled with a fluorochrome or enzyme to ensure more streamlined staining.
- Primary body class (isotope) and/or subclass: To verify compatibility and ensure effective binding and detection, it is important to know the class and/or subclass of the primary antibody you are using so you can choose a secondary antibody that is raised against the same subtype or subclass.
Consider these factors when choosing a secondary antibody for immunohistochemistry (IHC) experiments:
Factors Affecting Efficiency:
- Specificity: Determine the secondary antibody's ability to bind specifically to primary antibody-antigen complexes to minimize non-specific binding with random antigens and tissues and improve reproducibility of the results.
- Consistency: Choose secondary antibodies with low variability across different lots to maintain reproducibility and consistency in experimental results.
- Sensitivity: Select secondary antibodies with high sensitivity to detect antigens present in the tissue in low quantities to ensure stronger binding and easier recognition of epitopes in tissue samples.
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