CST BLOG: Lab Expectations

The official blog of Cell Signaling Technology® (CST) where we discuss what to expect from your time at the bench, share tips, tricks, and information.

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Pros and Cons of Different Multiplexing Techniques

Multiplex immunohistochemistry (mIHC) and multiplex immunofluorescence (mIF) are widely used to identify and localize different cell types within tissue samples. But choosing the right technique depends on the aims of the study and the tools available. A recent publication highlights the advantages and disadvantages of different approaches to mIHC/mIF and explains how these methods differ1.

Multiplex chromogenic IHC

Traditional chromogenic IHC uses DAB or AEC to visualize a single target on a whole slide. However, new chromogen substrates enable simultaneous or sequential multiplexing of 3-5 markers in as little as 10-15 hours. Advantages of chromogenic IHC are that it is affordable and relatively easy, as well as benefiting from established protocols and guidelines. It is also readily automatable for high throughput applications. Disadvantages are that most chromogenic substrates have a limited dynamic range (meaning marker intensity is, at best, semi-quantitative) and only very few can be combined for studying marker co-expression.

Multiplexed IHC consecutive staining on single slides (MICSSS)

MICSSS is similar to traditional chromogenic IHC but uses iterative cycles to visualize up to 10 markers on a whole slide2. Detecting one marker at a time eliminates the risk of steric hindrance or bleed-through that can compromise results. However, throughput is limited (each cycle takes 1-2 days to complete) and merging individual MICSSS images for analysis can be challenging. Additional drawbacks of MICSSS are that coverslip removal and chemical de-staining / antigen retrieval between cycles can damage tissue, while marker intensity is only semi-quantitative.

Multiplex IF

20-CAN-88567 Pros and Cons of Different Multiplex Techniques

Multiplex IF uses antibodies labeled with fluorophores to simultaneously detect multiple markers. Depending on the protocol, this can take from 2-20 hours. Standard IF microscopes typically allow whole slide visualization of 4-5 markers in a single round of staining. In contrast, multispectral microscopes are used to analyze up to 8 markers within distinct (0.66mm2) regions of interest (ROI) that can subsequently be tiled. Emerging studies suggest that a cyclic staining approach to multiplex IF may allow for detection of 30-60 markers3,4,5. A major advantage of multiplex IF is that the large linear dynamic range of most fluorophores allows for quantitation of marker intensity. However, fluorophores must be chosen carefully to prevent bleed-through and, where tyramide signal amplification (TSA) is used to boost signal intensity, additional checks are required to rule out potential blocking with TSA reagents.

Tissue-based mass spectrometry

By using primary antibodies labeled with metal tags, tissue-based mass spectrometry enables visualization of 40 or more markers in parallel, with staining possible in just 12 hours at 4oC. Like multiplex IF, it is used to analyze distinct (1mm2) ROIs and provides a quantitative measurement of marker intensity. This is achieved via two main approaches - multiplexed ion beam imaging by time of flight (MIBI™-TOF) and imaging cytometry (IMC) – which use distinct mechanisms to generate ions from each ROI prior to TOF analysis. Because tissue-based mass spectrometry avoids the use of fluorophores, the risk of signal fading, spectral overlap or autofluorescence is eliminated. Counterbalancing these advantages, the main drawbacks of tissue-based mass spectrometry are that instrumentation is extremely costly and extensive training is required.

Digital spatial profiling (DSP)

Digital spatial profiling is a relatively new technique that uses primary antibodies bound to UV-cleavable fluorescent DNA tags to quantify markers6. While ROIs are smaller (0.28mm2) compared to multispectral microscopy and tissue-based mass spectrometry, DSP can detect more markers (in practice 40-50, but theoretically as many as 800) in less time (1 hour) using just a single round of staining. The main limitation of DSP is that it does not produce an image. Instead, up to 4 fluorophore-labeled antibodies are used to select ROIs before the cleaved DNA tags are transferred to a multiwell plate for analysis.

Although each technique described here has its own advantages and disadvantages, the success of any of these methods depends on the use of highly specific antibodies, chosen to match your sample preparation method. For FFPE tissues, CST offers a catalog of IF-paraffin or IHC-validated antibodies, while for frozen tissues, consider selecting from our IF-frozen validated antibodies. Because we adhere to the Hallmarks of Antibody Validation - six complementary strategies that can be used to confirm the functionality, specificity and sensitivity of an antibody in any given assay – you can rely on us for mIHC/mIF results you can trust, whichever method you choose.


  • Taube, J.M. et al. (2020) J Immunother Cancer. 8 (1)
  • Remark, R. et al. (2016) Sci Immunol 1 (1)
  • Goltsev, Y. et al. (2018) Cell 174 (968–81)
  • Lin, J-R. et al. (2018) eLife 7.
  • Gerdes, M.J. et al. (2013) Proc Natl Acad Sci USA 110 (11982–7)
  • Merritt, C.R. et al. (2020) Nature Biotechnology 38 (586–599)

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