Pros and Cons of Different Multiplexing Techniques for IHC and IF

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 differ.¹

Multiplexed 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 three to five markers in as little as 10 to 15 hours. Advantages of chromogenic IHC include that it is affordable and relatively easy, as well as the fact that it leverages established protocols and guidelines. It is also readily automatable for high-throughput applications. Disadvantages include that most chromogenic substrates have a limited dynamic range (meaning marker intensity is, at best, semi-quantitative) and that 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 the technique uses iterative cycles to visualize up to 10 markers on a whole slide.² Detecting one marker at a time eliminates the risk of steric hindrance or bleed-through, which can compromise results. However, throughput is limited (each cycle takes one to two days to complete) and merging individual MICSSS images for analysis can be challenging.

Additional drawbacks of MICSSS include that coverslip removal and chemical de-staining / antigen retrieval between cycles can damage tissue, while marker intensity is only semi-quantitative.

Multiplex IF 

Multiplex IF uses antibodies labeled with fluorophores to simultaneously detect multiple markers. Depending on the protocol, this can take anywhere from two to 20 hours. Standard IF microscopes typically allow whole-slide visualization of four to five markers in a single round of staining. In contrast, multispectral microscopes are used to analyze up to eight 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 the detection of 30-60 markers.^3,4,5 A major advantage of multiplex IF is that the large linear dynamic range of most fluorophores allows for the 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.

Blog: Fluorescent Staining Using Multiple Antibodies: Two Common Techniques

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 4°C. 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), both of 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 markers.⁶ 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 four fluorophore-labeled antibodies are used to select ROIs before the cleaved DNA tags are transferred to a multi-well plate for analysis.

Antibody Selection for Multiplex Experiments 

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, Cell Signaling Technology 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.

Additional Resources: 

• Blog: Fluorescent Staining Using Multiple Antibodies: Two Common Techniques
• Blog: Demystifying Antibody Panel Design for Multiplex IHC
• Blog: Strategies for Successful Multiplex Immunofluorescence Experiments Using Conjugated Antibodies

References: 

1. Taube JM, Akturk G, Angelo M, et al. The Society for Immunotherapy of Cancer statement on best practices for multiplex immunohistochemistry (IHC) and immunofluorescence (IF) staining and validation [published correction appears in J Immunother Cancer. 2020 Jun;8(1):]. J Immunother Cancer. 2020;8(1):e000155. doi:10.1136/jitc-2019-000155
2. Remark R, Merghoub T, Grabe N, et al. In-depth tissue profiling using multiplexed immunohistochemical consecutive staining on single slide. Sci Immunol. 2016;1(1):aaf6925. doi:10.1126/sciimmunol.aaf6925
3. Goltsev Y, Samusik N, Kennedy-Darling J, et al. Deep Profiling of Mouse Splenic Architecture with CODEX Multiplexed Imaging. Cell. 2018;174(4):968-981.e15. doi:10.1016/j.cell.2018.07.010
4. Lin JR, Izar B, Wang S, et al. Highly multiplexed immunofluorescence imaging of human tissues and tumors using t-CyCIF and conventional optical microscopes. Elife. 2018;7:e31657. Published 2018 Jul 11. doi:10.7554/eLife.31657
5. Gerdes MJ, Sevinsky CJ, Sood A, et al. Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue. Proc Natl Acad Sci U S A. 2013;110(29):11982-11987. doi:10.1073/pnas.1300136110
6. Merritt CR, Ong GT, Church SE, et al. Multiplex digital spatial profiling of proteins and RNA in fixed tissue. Nat Biotechnol. 2020;38(5):586-599. doi:10.1038/s41587-020-0472-9