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Is your IHC staining not working? — Tips for selecting the optimal secondary antibody/signal amplification strategy

Oct 07, 2015
selecting secondary antibody and signal amplification reagents

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**

  • Consider using directly conjugated antibodies in IHC only for the detection of very abundant target proteins, such as beta-actin or alpha-tubulin.
  • For medium to low abundant proteins, secondary antibodies are a good detection choice and provide some level of signal amplification (multiple secondary antibodies can bind to a single primary antibody).
  • For very low abundant proteins, we suggest using biotinylated secondary antibodies in combination with conjugated avidin/streptavidin. One very common strategy is the Avidin-Biotin Complex (ABC) method that results in high signal amplification. This is due to a single avidin molecule being able to simultaneously bind up to four biotin molecules. The biotinylated secondary antibody functions as a bridge between the antigen bound primary antibody and large pre-formed avidin and biotinylated enzyme complexes. Upon substrate addition these enzyme complexes convert the substrate to produce the amplified signal (see image below). A pre-incubation step of avidin with the biotinylated enzyme is required to form the large complexes. Please note that this should be performed in a dedicated ratio to prevent avidin saturation.
ABC IHC detection method

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:

  • When selecting a fluorophore conjugated secondary antibody, ensure that your microscope is able to excite and detect the fluorophore appropriately. This can be done by determining the excitation and emission spectra of the fluorophore of interest and the lasers/filters included in your microscope.
  • Select photostable fluorophores such as Alexa Fluor® and DyLight Fluor® dyes rather than FITC and PE, which are highly susceptible to fading/photobleaching.
  • When performing experiments with multiple fluorescent labels, ensure that each fluorophore can be spectrally separated and that one fluorophore does not get detected in another fluorophore’s channel (a process known as bleed-through). We suggest that you mock up the fluorophore excitation and emission spectra with the help of a spectrum viewer at the experimental design stage.

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.

Browse our wide range of secondary antibodies and avidin/streptavidin products.

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.

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