Tips for Optional Steps 12 and 14 – Incubate with Secondary Antibody and/or Amplification Reagent


  • Especially for multiplexing experiments, it might be tempting to use directly conjugated primary antibodies to mitigate the risk of non-specific secondary antibody binding.  Although these antibodies are suitable for the detection of certain high abundant targets, we generally advise using secondary antibodies in IHC experiments. Multiple secondary antibodies bind to a single primary antibody, thereby leading to signal amplification

  • To reduce the risk of background staining from non-specific secondary antibody binding, select cross-adsorbed/pre-adsorbed secondary antibodies. Also, include a secondary only control in your experimental design to control for  this type of background

  • For very low abundant proteins, we suggest using biotinylated secondary antibodies in combination with conjugated avidin (to form an avidin-biotin complex; ABC). Since a single avidin molecule can simultaneously bind up to four biotin molecules, this method results in higher signal amplification. Labeled streptavidin is now commonly used as a substitute for avidin in the Labeled Streptavidin Biotin (LSAB) method (Ramos-Vara 2005). Signal amplification can also be achieved by using the Peroxidase Anti-Peroxidase (PAP) and Alkaline Phosphatase Anti-Alkaline Phosphatase (APAAP) methods (see IHC protocol 6)

  • When using biotinylated primary or secondary antibodies, ensure that you have blocked endogenous biotin prior to primary antibody incubation (see tips for step 6

  • For tissues rich in Fcγ receptors, consider using fragment secondary antibodies such as Rabbit F(ab’)2 Anti-Mouse IgG:HRP (STAR13B). This type of fragment antibodies lacks the Fc region, thereby mediating Fcγ receptor interactions

  • When selecting fluorophore conjugates, ensure that you are able to excite and detect the fluorophore optimally. Therefore, review the excitation and emission spectra of your fluorophore of interest as well as the lasers and filters of your microscope 

  • To reduce photobleaching (chemical destruction of a fluorescent dye), select photostable fluorophores belonging to new generation dyes such as Alexa Fluor and DyLight Fluor. Although still used in imaging applications, traditional dyes such as FITC are highly susceptible to fading/photobleaching and should therefore not be your first conjugate choice 

  • While spectral separation unmixing software has significantly advanced in recent years, for multiplexing experiments, we still recommend that you review the excitation and emission spectra of  fluorophores in advance to minimize spectral overlap (Lavdas no date). Selecting fluorophores with no or very little overlap minimizes the risk of one fluorophore getting detected in another fluorophore’s channel, a process commonly referred to as bleed-through, cross-talk or cross-over