Benefits and Drawbacks of Various Multiplexing Methods for IF and IHC
To locate and identify several cell types in tissue samples, multiplex immunofluorescence (mIF) and multiplex immunohistochemistry (mIHC) are often utilized techniques. However, the study’s objectives and the available resources will determine which approach is best. A new article describes the differences between several techniques to mIHC/mIF and lists their benefits and drawbacks. Read more about multiplex immunoflourescence for cancer by visiting our website and if you have any questions related to this topic, connect with us.
Chromogenic Multiplexed IHC
Conventional chromogenic IHC visualizes a single target on an entire slide using DAB or AEC. However, three to five markers may be multiplexed sequentially or simultaneously in as little as 10 to 15 hours thanks to novel chromogen substrates. Chromogenic IHC has the advantages of being somewhat simple and inexpensive, as well as utilizing well-established procedures and standards. Additionally, it is easily automatable for applications requiring high throughput. The fact that only a few number of chromogenic substrates can be coupled to research marker co-expression and that the majority have a narrow dynamic range (meaning marker intensity is, at best, semi-quantitative) are drawbacks.
Consecutive Multiplexed IHC Staining on Single Slides (MICSSS)
Similar to conventional chromogenic IHC, MICSSS visualizes up to 10 markers on a single slide using iterative cycles. Results may be compromised by bleed-through or steric hindrance, which is avoided by detecting one marker at a time. However, it might be difficult to combine separate MICSSS pictures for analysis, and throughput is constrained (each cycle takes one to two days to complete).
Other disadvantages of MICSSS include the fact that marker intensity is only semi-quantitative and that tissue injury may result during coverslip removal and chemical de-staining/antigen retrieval in between cycles.
Multiplex IF
Multiplex IF detects many indicators at once by using antibodies tagged with fluorophores. This might take two to twenty hours, depending on the procedure. Four to five markers may usually be seen on the whole slide in a single staining cycle using standard IF microscopes. On the other hand, up to eight markers are examined using multispectral microscopes inside discrete (0.66mm2) regions of interest (ROI), which may then be tiled.
According to recent research, multiplex IF using a cyclic staining method could be able to identify 30–60 markers. The ability to quantify marker intensity because to the wide linear dynamic range of the majority of fluorophores is a significant benefit of multiplex IF. To avoid bleed-through, fluorophores must be carefully selected. If tyramide signal amplification (TSA) is used to increase signal strength, further testing is necessary to rule out any blocking with TSA reagents.
Mass spectrometry based on tissues
Tissue-based mass spectrometry allows the visualization of 40 or more markers in parallel utilizing primary antibodies tagged with metal tags, and staining may be completed in as little as 12 hours at 4°C. Similar to multiplex IF, it offers a quantitative assessment of marker intensity and is employed to examine discrete (1mm2) ROIs. This is accomplished using two primary methods: imaging cytometry (IMC) and multiplexed ion beam imaging by time of flight (MIBI-TOF), which both create ions from each ROI using different processes before TOF analysis. There is no chance of signal fading, spectrum overlap, or autofluorescence using tissue-based mass spectrometry as fluorophores are not used. Notwithstanding these benefits, tissue-based mass spectrometry’s primary disadvantages are the high cost of the equipment and the need for intensive training.
DSP, or digital spatial profiling
A relatively recent method for quantifying markers is digital spatial profiling, which employs primary antibodies attached to UV-cleavable fluorescent DNA tags. Although ROIs are smaller (0.28mm2) than those of tissue-based mass spectrometry and multispectral microscopy, DSP can identify more markers (40–50 in reality, but up to 800 in theory) in less time (1 hour) with only one staining cycle. DSP’s primary drawback is its inability to generate images. Rather, the cleaved DNA tags are moved to a multi-well plate for examination after ROIs have been selected using up to four fluorophore-labeled antibodies.
Choosing Antibodies for Multiplex Tests
The success of each of the techniques discussed here depends on the employment of highly specific antibodies that are selected in accordance with your sample preparation process, even if each approach has pros and cons of its own. Consider choosing from our IF-frozen validated antibodies for frozen tissues, or use Cell Signaling Technology’s catalog of IF-paraffin or IHC-validated antibodies for FFPE tissues.
You can trust us for reliable mIHC/mIF findings using whatever technique you select since we follow the Hallmarks of Antibody Validation, which are six complimentary methodologies that may be utilized to confirm the functionality, specificity, and sensitivity of an antibody in any particular assay.