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IHC Tip 6 - Amplify as there is no tomorrow

Aug 12, 2015

Tips for the incubation with secondary antibody & amplification reagent step

  • We recommend the use of directly conjugated antibodies in IHC only for the detection of very abundant target proteins (e.g. beta-actin and alpha-tubulin). 
  • For medium to low abundant proteins, we recommend using secondary antibodies for detection. We make this recommendation due to the fact that multiple secondary antibodies bind to a single primary antibody thereby leading to amplification of the signal.
  • For very low abundant proteins, we recommend using biotinylated secondary antibodies in combination with conjugated avidin/streptavidin,  Neutralite Avidin, and  Neutralite Avidin:Cy5 . We make this recommendation due to the high signal amplification of this method, which is due to a single avidin molecule being able to simultaneously bind up to four biotin molecules. 
  • When using biotinylated antibodies, ensure to block endogenous biotin prior to primary antibody incubation. For this specific purpose Bio-Rad offers a ready-to-use avidin-biotin blocking system.
  • For your convenience, we also offer the Histar Detection kit series, STAR3000, which provides linking and labeling reagents for the visualization of cellular antigens in tissue specimens. The Histar detection kits are biotin-free systems based on a novel polymer-based labeling technology. 
  • 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 looking up the excitation and emission data 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). For this purpose we recommend to mock up the fluorophore excitation and emission spectra with the help of a spectrum viewer at the experimental design stage.

View all secondary antibodies 

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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