In this blog post, we focus on selecting secondary antibody and signal amplification reagents, and provide some tips to help you nail this part of the IHC design process. Visit our IHC tips page for more immunohistochemistry tips.
In order to choose the best detection system, some basic information on the relative expression levels of your target antigen is required, as the ideal strategy will depend on whether the target antigen is of high, medium or low abundance.
**Bonus tip- The presence of endogenous biotin in some tissues can lead to high, non-specific background staining when using biotinylated antibodies. Therefore, be sure to block endogenous biotin prior to the primary antibody incubation step. Bio-Rad offers a ready-to-use avidin-biotin blocking system for this purpose**
Overview of the ABC IHC detection method
Although the ABC method provides significant amplification, it has limitations that prevent its application for certain analyses. For example, if you are interested in simultaneous detection of several antigens in one tissue specimen, a technique known as multiplexing, a fluorescence based strategy may better suit your needs. Although multiplexing can be performed using multiple enzymatic labels it is more restrictive due to the small number of precipitate colors (a result of the chromogen and substrate reaction), co-staining limitations, and the inability to quantitate antigen expression. For example, when visualization of two antigens located in close proximity or the same cellular compartment is desired, one precipitate might stain the entire compartment making detection of the second antigen with a different chromogen impossible. Fluorescent IHC addresses these limitations and may therefore be more applicable to your research goals. However, there are a few things you should keep in mind when selecting fluorophores for your IHC experiment:
For all IHC experiments, the use of proper controls is critical in order to ensure the accuracy of your staining. Visit our IHC page for a thorough overview of appropriate controls for your IHC experiment.
Alexa Fluor® is a registered trademark of Molecular Probes Inc. OR, USA.
DyLight® Fluor is a trademark of Thermo Fisher Scientific Inc. and its subsidiaries.