Published January 30, 2019

Multiplex IHC results in one work day? Optimized workflows make it happen!

Conventional immunohistochemistry (IHC) methods only provide insight into a limited number of markers of interest, prompting the need for development of multiplexed methods to assess and characterize multiple cell phenotypes and their spatial context in a single sample.

Until recently, there has been a trade-off between technology platforms that enable high multiplexing for the research and detection of many biomarkers and those that preserve the morphology of the sample with a low level of plex. Ultivue UltiMapper™ I/O kits using InSituPlex® staining technology for highly multiplexed in situ imaging bridge this technology gap, enabling high-content tissue analysis while providing an integrated workflow using conventional autostainers, imaging scanners, and analysis software. This seamless integration to existing histology workflows enables users to go from staining to analysis within the same day.

The InSituPlex DNA-barcoding staining technology is what drives this accelerated workflow to enable so many benefits for mIHC:

  • High Multiplexing In Situ: High number of markers with whole slide imaging
  • Sample Fully Preserved: Complete retention of sample antigenicity
  • Plug-in Implementation: Validated on multiple tissue types, using widely available fluorescence microscopes
  • Improved Workflow: Single staining and simultaneous amplification for all markers lowers sample-to-answer time
  • Versatile Implementation: Same technology, same reagents from research to clinic: equally applicable from discovery to downstream high throughput testing


So how do we go from staining to analysis in one day? Let’s take a look. There are three components to standard histological workflows: Staining, Imaging, and Image Analysis. Here’s a rough timeline and overview of the process.

Ultivue Workflow


Ultivue is optimized to run on the BOND RX from Leica Biosystems. This means that all you have to do is load your slides onto the tray, download the protocol and press start. You can antigen retrieve and stain 30 slides on the BOND in around 5.5 hours.

After staining, the slides need to be imaged. Whole-slide imaging can be done on most scanner systems with the correct filter configurations, and requires no spectral unmixing. We have found the ZEISS Axio Scan Z1 to be the fastest imaging system, with a throughput of around 10-12 minutes per slide. This means that you can scan 10 slides in around 2 hours.

Once the slides are scanned (around 7.5-8 hours later), they are ready for whole-slide analysis using commercial software solutions like HALO from Indica Labs. The whole-slide analysis allows for unbiased characterization of single-cell phenotypes over entire tissue sections.

Summing it all up, the InSituPlex technology and UltiMapper kits dramatically reduce the time it takes for researchers to get results from their tissue samples. Assay kits come ready with an optimized protocol with a single staining step for all targets and a simultaneous amplification step to enhance detection sensitivity on low-expressing targets. Moreover, the assay is optimized for all panel targets on multiple tissue types, eliminating the months-long process of assay optimization or re-optimization for new tissue samples. Workflows can be fully automated and integrated into standard IHC labs, enabling high-throughput and reproducible data without the need for unique instrumentation. Samples can be imaged using a standard multi-color fluorescence imaging system, without the need for spectral unmixing. This enables researchers to spend time more time analyzing results, not optimizing assays.

For more information on our workflow and technology applications, download our complete collection of 6 posters that were presented at SITC 2018.

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