Multi-Antibody Immunofluorescence Available

Related Core Facilities:

Tuesday 11/12/2013

An example of multi-color immunofluorescence using 3 mouse antibodies on mouse tissue. Beta Catenin in red, smooth muscle actin in white and HHF35 in green with a DAPI counterstain on mouse intestine.It was once widely believed that immunofluorescence could not be successfully done on formalin-fixed, paraffin-embedded (FFPE) tissue because of the high level of autofluorescence. In the past several years, however, new fluorochromes and detection strategies have made it possible to not only stain FFPE tissue but produce superior images from these samples. Formalin-fixed samples provide the best morphology and stability of antigen expression.

The staff in Experimental Histopathology has taken these new reagents and techniques even further to establish protocols that allow for bright and specific multi-color immunofluorescence on FFPE samples.

The core has expertise in:

  • Protocols to stain with 2 and 3 antibodies on the same section.
  • Using 2-3 antibodies made in the same host with no crossover detection
  • Using more than one mouse monoclonal antibody on mouse tissue (see below)
  • Amplification of weak signal above the level of autofluorescence

Please keep in mind that multi-antibody staining does require additional optimization steps to ensure that detection reagents are not crossing over and that the staining intensities are compatible. These protocols also require additional controls for accurate interpretation and analysis.

 

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Shared Resources are core facilities that provide services and access to specialized equipment for research activities.


Rajesh K. Uthamanthil, DVM, PhD, DACLAM

Associate Vice President, Shared Resources