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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.