Next generation IHC techniques

Labeled monoclonal antibodies analyzed by mass spectrometry allow for quantitative multiplexing on intact tissue samples.

by Luca Benfatti

Recent advances in immunohistochemistry (IHC) techniques have improved their dynamic range by multiple orders of magnitude. This significant jump in the quantity of proteins detectable on a tissue sample is truly the next generation of IHC.

The dynamic range of IHC is 1 log and 2.5 logs for quantitative immunofluorescence (QIF), which measures the relative amount of a target protein in a tissue sample (1). Mass spectrometry IHC (MSIHC) can reach a value of 5 logs.

Currently, there are limitations to IHC due to the restriction of detection techniques, loss of spatial resolution with chromogenic methods, loss of sensitivity with fluorescent methods, heterogeneity and variability in pre-processing of samples.

With the ability to allow multiplexed and directly quantitative imaging of tissue samples, this technique helps to overcome many of the current IHC limitations. It is indeed cutting-edge technology for basic and clinical research.

Coupling mass spectrometry with immunohistochemical analysis: laser vs. ion beam


Mass cytometry has the ability to measure multiple genes in the same cell to define their function. Target specific primary antibodies are labeled with rare lanthanide metals with high-quality mass that is easily distinguishable by the mass cytometer. These antibodies are incubated with the tissue samples in a similar way to the IHC protocol.

A key aspect is the need to liberate the heavy metal ions for separation and quantitation with minimal overlap. The amount and number of heavy metals detected is proportional to the amount and number of proteins bound by the labeled antibodies. The instrument can analyze >1000 cells/spost-translationaland this can be applied to a normal IHC staining experiment.

Two research groups have coupled these techniques in slightly different ways. Bernd Bodenmiller's group uses Labeled CyTOF® instrument (flow cytometry modified for detecting mass tagged antibodies and assessed by time-of-flight mass spectrometry) which utilizes a laser to destroy the tissue/antibodies and free heavy metal ions at a resolution of 1 μM. An analyzed image is created which looks very similar to a routine IHC but with quantitative multiplexed information (2).

Garry Nolan’s group uses a scanning ion beam to liberate the ions, which improves the resolution (smaller amounts vaporized at a time for higher cellular clarity) but requires more specialized setup (vacuum, multiple detector MS). This method is called multiplexed ion beam imaging (MIBI) (3). Both groups use imaging software to reconstruct the 2-D stained tissue image from the detected heavy metal ions.


The great advantage of this technique is that multiple monoclonal antibodies for the same protein are analyzed, revealing antibody specificity by directly comparing one antibody to another. It highlights the different specificity of the antibodies for different protein domains as a function of changes in the structure of the protein or post-translational modifications.

The technique also has the advantage of allowing subtle changes in multiple proteins to be studied at once, due to the simultaneous use of a high number of heavy metal ions (high plexing capability). Finally, the multiplex advantage allows the use of antibodies against housekeeping proteins for reference and normalization, giving detailed pre-processing information and providing more reproducible and accurate IHC results. This opens the door to IHC as a more trustworthy clinical and diagnostic application.

High-quality IHC validated antibodies needed

It is important to note that for both methods the quality of the antibody is crucial. Poorly characterized or cross-reactive antibodies will give non-reproducible results because the interaction between antigen and antibody still has the primary role in the measurement of expression.

We offer RabMAb® products; a range of high-quality rabbit monoclonal antibodies that complements this innovative technology. RabMAb products are ideal IHC reagents:

Additionally, we provide high quality rabbit monoclonal antibodies for anatomic pathology (IVD IHC usage).

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