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Tips & Tricks to Multiplexing: Top 5 Reasons to Multiplex and Chromogenic versus Immunofluorescent Detection

Rhian Evans
Rhian Evans Ph.D., Scientist

Multiplex staining is an advancement on the single marker per tissue section immunohistochemistry technique, enabling the detection of multiple markers simultaneously on the SAME tissue sample. Watch our 2 short videos below to learn more about multiplexing! Download the complementary infographics to keep the points on hand when you need to revisit the information.

Top 5 Reasons to Multiplex

Check out and learn with Larry from Leica Biosystems about the top 5 reasons to implement multiplex staining into your tissue-based research.

To use multiplex staining in your research project

Multiplex advanced staining can provide biological information that helps to better understand the molecular mechanisms of health and disease for biomarker and drug discovery.

1. Conserve tissue resources

Multiplexing allows multiple immunohistochemistry or immunofluorescent tests to be performed on the same sample, maximizing the amount of data obtainable.

2. Provide greater insights

Multiplex staining in tissue-based research provides insights into disease and patient heterogeneity by granting single slide visibility of the tumor microenvironment

3. Visualize protein and RNA

Multiplexing can stain for protein and DNA/RNA on the same tissue section, providing a detailed map to explore the relationship between protein and gene expression.

4. Reduce laboratory time

Automated research stainers can reduce hands-on time and turnaround time to fully stained slides compared to manual methods, and can improve the reproducibility of results by managing incubation time, temperature, and reagent application.

5. Analyze with digital pathology and AI

Digital pathology enables the visualization of stained tissue sections on a computer screen and can be used with quantitative image analysis applications for more detailed outputs.

Chromogenic vs. Immunofluorescent Multiplex Staining

Unsure of whether to use chromogenic or immunofluorescent multiplex detection?
Larry from Leica Biosystems showcases the advantages and disadvantages of both types of detection.

When adopting multiplex staining for tissue-based research, it is important to consider whether to use chromomogenic or immunofluorescent detection.

Here are key reasons to use either detection system.

Chromogenic Multiplexing

  1. Chromogenic multiplex detection is most useful for determining a positive (present) or negative (absent) result.
  2. Chromogenic multiplex assays are best for longevity as it produces permanently stained slides that are resistant to photobleaching
  3. Chromogenic staining systems can be viewed and digitized with readily available brightfield microscopes and scanners.
  4. Chromogenic stains can be more sensitive based on the detection systems used to amplify the signal of the primary antibody

The disadvantages of chromogenic multiplex detection:

  • It is difficult to resolve between multiple colocalized markers as it has a lower dynamic range for quantitation.
  • More assay optimization is required to find the best chromogen color combination, order, and stability for additional assay steps.

Immunofluorescent Multiplexing

  1. Immunofluorescent multiplex detection can be used for larger panels of markers, including a number of colocalized targets as multispectral imaging systems enable layer separation.
  2. Immunofluorescent multiplex assays have a higher dynamic range, so they can be more useful for quantitation between high and low abundance proteins.
  3. Multispectral imaging systems for immunofluorescence can be used to analyze layers independently, then merged back together for comprehensive image analysis.

The disadvantages of immunofluorescent multiplex detection:

  • Requires more complex and expensive imaging and scanning systems.
  • Slides produced are susceptible to photobleaching and stains are not permanent.

About the presenter

Rhian Evans
Rhian Evans , Ph.D., Scientist

Rhian is a Scientist from Swansea University in Medical and Healthcare Studies and was featured in several collaborative publications. Rhian’s research-based background focused on tissue-based pathology in Multiple Sclerosis, primarily using immunohistochemical analysis and in vitro molecular techniques. She spent a short period conducting routine PCR testing for COVID-19 at the end of 2020.

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