Immunohistochemistry (IHC) Resources

Multiplexing Resources

ihc multiplexed slide image
What is Multiplexed Immunohistochemistry?

Multiplex immunohistochemistry (mIHC) enables the detection of multiple biomarker antigens on the same tissue section. Compared to single stains, simultaneous biomarker detection enables a greater understanding of the tissue. The scientific questions that can be addressed by mIHC are many, and span clinical, translational, and basic research applications. 

For example, biomarkers for cell lineages show the spatial organization of different cell types within the structural context of a complex tissue.  The components of the immune system within the tumor microenvironment,  the dynamics of wound healing, or a developing tissue can be better understood.  With translucent chromogens (link to chromogen page), co-localization of proteins within subcelleular compartments can be determined. In short, mIF assays has the advantage of producing better information about the tissue and its cellular and protein organization, while using less of precious samples.


Multiplex IHC can provide unique biological insights that enable investigators to build hypotheses not possible with single stains!

IHC melanoma slide
number one

Here is an example of immune cells infiltrating a melanoma  tumor. This melanoma specimen was stained using a chromogenic triplex assay in which LAG3, CD8, and PD-L1 biomarkers can be visualized on the same slide.  Here, CD8-positive cytotoxic T lymphocytes (stained with a yellow chromogen) are effectively excluded from the tumor parenchyma.

number two

This T cell exclusion is associated with the expression of the checkpoint protein PD-L1 (stained with a purple chromogen) in a thin layer of stromal cells that surround the tumor parenchyma.  It is possible this wall of PD-L1 serves to inhibit T cell activation leading to the exclusion of CD8-positive cells from tumor parenchyma.

number three

The presence of the inhibitory co-receptor LAG3 (stained with a silver black chromogen) in those CD8-positive T lymphocytes that are closest to the wall of PD-L1-expressing stromal cells is consistent this hypothesis.  LAG3 expression in the CD8-positive cells closest to PD-L1 could signal a state of exhaustion in those T cells.

How to get started with Multiplexed Immunohistochemistry

Successful mIHC needs careful design and optimization, 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.

Discovery Interactive Assay Design Tool screenshot
Explore the DISCOVERY Interactive Assay Design Tool

This tool demonstrates some of the applications available on the Ventana DISCOVERY ULTRA, and gives you the ability to create and save a variety of assay types and generate associated product lists.

Key considerations for a successful multiplexed IHC assay

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 optimizing 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 customized to better suit your research needs when incorporating stable tyramide chromogens.

recommended chromogen sequence

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 (link to chromogen page), 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.
TSA multiplexing image
multiplexing protocols

Interested in trying chromogenic multiplexing? 

These example protocols are a good place to start! Example protocols provide antibody and chromogen incubation times and other details; however you may need to adjust the protocol to your own antibodies and tissues.

A CD3 and CD8 Chromogenic Duplex Protocol

A Chromogenic IHC 3-plex on Breast Carcinoma

A Chromogenic IHC 4-Plex on Prostate Carcinoma

A Chromogenic IHC 5-Plex on Tonsil

A Chromogenic IHC 6-Plex on Tonsil

Co-localization 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 color 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. Color combinations obtained from translucent chromogens include a purple-yellow color shift to a fiery red, a yellow-teal color shift to a mild green, and a purple-teal color shift to dark indigo blue.

Co-localization IHC Staining

Controls for Multiplexing IHC

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 optimization.  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

Want to detect RNA in FFPE tissues or perform multiplexed same-slide ISH and ICH for nucleic acid and protein multiomic studies?

Learn more about RNA detection using HCR™ probes from Molecular Instruments.

Multiplexed IHC FAQs

Multiplexed IHC FAQs

Yes, our next-generation chromogens are deposited using TSA deposition so the chromogen signal is covalently bound.  This allows mild denaturing conditions to wash off the first primary without effecting signal, and a second primary of the same species can be used in the following step.

No, our translucent chromogens allow for co-expression and co-localization without the need for a fluorescent microscope.  Further, brightfield also shows hematoxylin counterstain so that the entire tissue context can be seen - something not possible with fluorescence.

The rule of thumb is DAB > purple> red > yellow > teal > green > silver. However, localization of each target, sensitivity of each biomarker epitope, and chromogen translucence must also be considered.

Yes, stain frozen sections in the same way as you would FFPE but select 'Wet Slide Load' when building the protocol to disable the deparaffinization step. You may not require any further retrieval of the epitope.

Include a denaturation step to quench any residual AP between steps.


Multiplexed IHC Resources

Download this brochure for more information on chromogenic and fluorescent IHC multiplexing.

This white paper will help you to get started with IHC multiplexing.

Download this presentation for amazing examples of chromogenic and fluorescent 2-through 5-plexes.

DISCOVERY instruments and reagents are for research use only. Not for diagnostic purposes.