Immunochemistry

Location: Thomas Building, DE-360
Contact phone: (206) 667-6166
Contact fax: (206) 667-6845
Contact e-mail: exphisto@fredhutch.org

Experimental histopathology offers extensive immunohistochemistry services. (See our specialized services, multi-antibody immunofluorescence and mouse-on-mouse staining below.) The laboratory uses automated immunochemistry stainers which offer consistency and standardized protocols on large and small scale projects.

Antibodies can be provided by the user or Experimental Histopathology laboratory. We have over 100 hundred antibody protocols worked up for a number of species. Please contact the lab for an updated list of antibodies.

We can use various detection methods including immunoperoxidase, alkaline phosphatase, and fluorescence. Protocols include dual IHC and multi-color IF. The staff has a great deal of experience with antigen retrieval techniques for formalin fixed tissue and with staining human and animal tissue.

Multi-Antibody Immunofluorescence on formalin-fixed, paraffin-embedded samples

The staff in Experimental Histopathology has 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.

Mouse-on-Mouse Staining

Special care must be taken when using mouse monoclonal antibodies in mouse tissue to ensure that the reagents used to detect the mouse monoclonal antibodies are not also reacting with endogenous antibodies in the mouse tissue. This is a concern even with immunocompromised mice as even some strains of NSG mice have low level expression of immunoglobulin.

The staff in Experimental Histopathology has adapted a system for mouse-on-mouse staining to provide maximum flexibility and include different chromagens, fluorochromes, and multi-antibody staining protocols while maintaining cost effectiveness. See our 2014 JHC publication on this technique

Save time and money - do not buy antibodies until you have consulted the lab!

Tips

  • Many antibodies work in formalin fixed tissue, however, some do not. The staff can help you determine if you should fix your tissue and help you choose the best fixative.
  • Many antibodies are not suitable for immunochemistry. The staff can help you identify the best antibody for your application.
  • We may already have an immunochemistry protocol worked up for your protein of interest. See our list of antibodies.

Doing Business with Experimental Histopathology

Thomas Building, DE-360
(206) 667-6166
exphisto@fredhutch.org

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