Immunohistochemistry Multiplexing

Multiplex immunohistochemistry, sometimes referred to as ‘multiple immunolabeling’ or ‘multiplex immunostaining’, is an advanced version of immunohistochemistry that allows researchers to acquire more information from the same sample.

Multiplex immunohistochemistry is commonly used in the examination of the arrangement of proteins and how they interact within neighbouring proteins.

Multiplexing Resources

View additional information and resources to understand best practices and applications of multiplexing:




Multiplexing FAQs

  • mIF requires high quality IHC validated primary antibodies, HRP-conjugated secondary antibodies, tyramide detection fluorophores, and an imaging system capable of resolving the wavelengths of the fluorophores being used in the assay.

  • Primary antibodies of any host species can be used in mIF, even if the antibodies are from the same host species. It is advisable to begin developing mIF with highly validated IHC antibodies. Fortis Life Sciences offers an extensive portfolio of IHC validated antibodies. Antibodies from other companies are compatible with Fortis antibodies but will need to be validated by the researcher.

  • The most common mIF sample is formalin fixed paraffin embedded tissue (FFPE). Since mIF with tyramide detection uses multiple rounds of heat induced epitope retrieval, non-FFPE tissue tends to degrade during the assay.

  • Fortis antibodies are validated for mIF with the Opal detection system from Akoya Biosciences. Use of other tyramide reagents will require independent validation by the researcher.

  • The mIF protocol outlines the basic optimization parameters. The recommendations given for mIF applications are starting points for assay development. Further optimization of antibodies will be needed using your own tissue. Differences in tissue fixation, epitope retrieval buffer, instruments used for epitope retrieval, and technical skill can impact the mIF assay. The mIF protocol outlines the basic optimization parameters.

  • Order of staining can impact the detection of antigens. Multiple rounds of epitope retrieval can destroy some epitopes. In this case, antibodies recognizing these markers need to be used early in the staining order. Fortis validated mIF panels will have established an initial staining order. This order should be confirmed on your own tissue before proceeding mIF studies.

  • Fluorophore pairings should be carefully considered when staining for markers that occur in the same cell type, especially if those markers are localized to the same subcellular location. In these cases, it is advised to use fluorophores whose spectra are non-overlapping.

  • Fortis uses steamers for HIER because this method is widely used. Other methods, such as microwave treatment, may be used but will require independent validation by the researcher.

  • The number of targets detectable is dependent on available tryramide fluorphores and the imaging system. Several slide scanners such as the Vectra Polaris offer the capability to perform a 9-color scan (8 immunostained markers + DAPI).