Multiplexing Resources

Successful mIHC needs careful design and optimisation, but is not as daunting as it may first appear.  Decisions will need to be made for the antigen retrieval approach, order of antibody application, detection chemistry and choice of chromogen for each epitope. However, with supportive education, reliable processes, and stable detection chemistry, we can help you avoid common implementation challenges and enable you to advance your research in the ways you imagined.

Antibody Selection

Traditional IHC multiplexing methods use different primary antibody species for each epitope on a slide to maintain signal specificity. Alternatively, directly labeled antibodies of the same species can be used together on the same slide to circumvent the need for different species primaries, but this can come at the expense of sensitivity. The use of TSA chemistry for chromogen or fluorophore deposition solves the problem of needing a different species primary for each step.

Antibody Order

A general rule for optimising a multiplex assay is to stain the lowest expressing, or most labile epitope first for optimal target detection.  Ultimately, low expressing and/or sensitive epitopes should be targeted early in the staining sequence while high expressing and stable epitopes should be targeted later in the staining sequence.  Targets requiring unique retrieval, blocking, and/or amplification reagents should be placed later in the staining sequence as those reagents could impact efficient binding of downstream targets.

Chromogen Order and Selection

When using multiple chromogens, order is important for obtaining the most distinct and vibrant hue for each chromogen. Our preferred chromogen order is DAB > purple > red > yellow > teal > green > silver. However, this is only a recommendation and the chromogen order can be customised to better suit your research needs when incorporating stable tyramide chromogens.

Tyramide Signal Amplification (TSA)

Tyramide signal amplification uses horseradish peroxidase (HRP)-conjugated secondary antibodies to form a covalent bond between the TSA-enabled chromogens or fluorophores and endogenous tyrosines in the tissue. The use of tyramide signal amplification mIHC provides advantages over traditional methods that work by precipitation. 

• TSA enables greater sensitivity: Amplification results from large quantities of chromogen or fluorophore being deposited at the site of the protein of interest, and results in an intense signal. This is especially useful for detection of low-abundance proteins.
• Use same-species primary antibodies: Using Ventana next-generation chromogens, which employ tyramide signal amplification, the multi-species limitation is eliminated. Because the chromogen or fluorophore is deposited with covalent bonds, each primary antibody can be denatured/stripped off before the application of another primary in the next step without compromising previously deposited chromogens. This eliminates the troublesome need to choose a primary antibody of a different species for each step of the multiplex.

Co-localisation IHC Staining

For target proteins within the same cell and the same cellular compartment (e.g., membrane, nucleus, cytoplasm) detection is ideally accomplished with translucent chromogens, such as purple, yellow and teal. These provide the advantage of producing a new colour when co-expressed. Stains such as DAB or silver should not be used for co-expression as these chromogens will likely obscure co-localized chromogens. Colour combinations obtained from translucent chromogens include a purple-yellow colour shift to a fiery red, a yellow-teal colour shift to a mild green, and a purple-teal colour shift to dark indigo blue.

Controls

For each cycle of staining, mild heat and chemical denaturing steps strip off the antibody complexes between while maintaining robust staining from previously deposited chromogens.  Some epitopes may lose antigenicity during these steps. Proper controls confirming epitope stability and efficient elution of primary-secondary antibody complex should be included during optimisation.  We recommend:

• Step 1 - Single stains for optimal antigen retrieval and antibody conditions
• Step 2 - Denaturing controls for determining antibody order
• Step 3 - Stripping controls for verification of signal specificity