Over the last few years, more and more researchers have embraced multiplex immunofluorescence (mIF), and for good reason. The ability to visualize many biomarkers in a single tissue section can transform a standard immunostaining experiment into a deep dive on cellular neighborhoods, signaling pathways, and immune infiltration. The CellScape™ Precise Spatial Proteomics platform is a popular choice for mIF, as the instrument leverages EpicIF™ (enhanced photobleaching in cyclic immunofluorescence) technology, a powerful workflow to achieve highly multiplexed protein detection quickly and easily.
While ready-to-use kits for the CellScape platform are available for jumpstarting data generation, often the most relevant assays for your unique research must be custom-designed. Here, we share some key considerations and best practices for developing and optimizing custom assay panels for your spatial proteomics research.
Define Clear Biological Goals
Begin with a well-defined question: Are you profiling immune cell populations in the tumor microenvironment, or exploring spatial heterogeneity in a specific signaling pathway? Your biological hypothesis will guide which markers to optimize first. With the CellScape platform, samples can be stored and revisited later, allowing you to expand your assay as new data emerge. This flexibility reduces the pressure to include every marker upfront and makes it easier to refine your approach over time without starting from scratch.
Antibody Selection
Choosing the right antibodies is essential for specific and reproducible staining. Consider the following best practices:
- To identify candidates for CellScape-compatible antibodies, look at commercially available clones that are validated for performance in immunofluorescence (IF) or immunohistochemistry (IHC) assays. Consult vendor datasheets to confirm specificity for each target.
- Choose clones with fluorophores compatible with EpicIF technology, which includes most commonly-used organic dyes in the Rhodamine, Cyanine, and BODIPY families.
- Use primary antibodies if possible, as they offer the simplest workflow and minimize the risk of cross-reactivity. However, if a compatible fluorescent primary antibody isn’t available for your target, a primary + secondary approach can be used instead. Custom conjugation is also an option to create CellScape-compatible primaries, with easy-to-use antibody labeling kits available from multiple vendors.
Balancing Fluorophores
Ideally, clone selection is completed in parallel with fluorophore selection. When building a multiplex panel, choose your fluorophores thoughtfully to maximize the number of markers detected in a single cycle. Up to four antibodies can be used in each cycle if the dyes do not have spectral overlap. To minimize issues with overlap, we recommend using the 5 filter sets built into the CellScape platform in the following combinations, depending on how many markers are included in each cycle:
| FS488 | FS488 |
| FS532 | |
| FS555 | |
| FS594 | |
| FS647 | FS647 |
Additional considerations for fluorophore selection include:
- Assign the brightest fluorophores to lower-expressed targets to maximize detection sensitivity.
- Prioritize biomarkers with limited fluorophore options first, then build your multiplex assay around them using targets with more dye-clone flexibility.
- Remember that EpicIF technology is not compatible with fluorescent proteins (GFP, RFP, YFP, etc) or quantum dots (qdots). Signals from these dye families cannot be removed with EpicIF solution.
Individual Antibody Optimization
Once selected, optimize and validate each antibody individually, with the goal of obtaining consistent and robust signal.
- Titrate antibodies in single-plex to determine the optimal concentration that provides strong signal without excessive background. Start with manufacturer-recommended concentrations, then systematically dilute to find the “sweet spot” that balances signal strength and background noise.
- Validate each conjugated antibody in a known positive control tissue and confirm the absence of off-target binding using negative controls or isotype controls.
- If bright artifacts are found on tissues, as shown in the image below, it is likely due to antibody aggregation. As with IF/IHC, this problem can be solved by centrifugation or filtration (using a low-protein-binding filter) of working antibody solutions.
- Optimize EpicIF photobleach times to ensure complete signal erasure. Signal from most antibody-fluorophore conjugates can be removed by 5-10 seconds of filtered photobleaching.
Iterative Refinement of the Multiplex Panel
Start Small and Expand
Start multiplexing with a manageable subset (4-6 markers). Run a pilot CellScape protocol and analyze signal:noise and specificity, adjusting titrations of each antibody if needed. Once the initial panel is established and validated, add new markers in groups of 4-6.
Consider Staining Order
Because of the gentle nature of the EpicIF workflow and filtered photobleaching, we have observed very few instances of markers that are harder to detect in late rounds of staining. However, any concerns about either steric hindrance or unstable epitopes can be mitigated by putting “high risk” markers in the earliest cycles.
Return to Antibody Selection and Fluorophore Balancing as Needed
Sometimes antibodies in multiplex do not perform as well as they do individually. It is normal for assay optimization to be a back-and-forth between individual antibody/fluorophore selection and multiplex validation. If an antibody doesn’t perform well in a panel, return to an earlier stage and choose an alternative antibody or adjust the titration to improve signal.
DNA Staining
DNA staining is critical for accurate cell segmentation. We recommend using SYTOX® dyes, as they are available in a variety of wavelengths that are compatible with EpicIF technology and the filters used on the CellScape platform. As with antibodies, the DNA dye should be titrated to optimize brightness and maximize signal:noise. There are a few considerations for optimal dye use:
- SYTOX dyes typically require more dilute concentrations than antibodies for optimal staining (50 – 250 pM range).
- High-affinity nucleic acid dyes typically require shorter incubation times compared to antibodies. Test different incubation and photobleach times to fine-tune the results.
- Nucleic acid stains can be retained by low-protein-binding filters. If a DNA dye is included in the same working solution as antibodies, either add the DNA stain after filtration or use centrifugation to clear antibody aggregates.
Manage Autofluorescence
Certain tissues (brain, for example) naturally have a high level of autofluorescence that can interfere with detection of fluorophores. Unique to the CellScape platform, the filtered photobleaching signal erasure step can also be utilized to decrease background autofluorescence. Increasing the timing of filtered photobleaching or adding an initial photobleach step before the first staining cycle can help mitigate issues arising from too much background signal.
Final Thoughts
Building a multiplex assay panel using primary-conjugated antibodies on the CellScape platform is simple and straightforward with the right methodical approach that harmonizes smart target selection, careful validation, and strategic experimental design. By leveraging both our VistaPlex Assay Kits and our best practices for assay design, you can easily create panels targeted for your unique research while maintaining reproducibility for trustworthy results.
References
- Exploring uncharted spatial biology with ChipCytometry highly multiplexed imaging
- Approachable, Agile, and Precise Spatial Biology with the CellScape Platform
- Highly multiplexed immunofluorescence imaging of human tissues and tumors using t-CyCIF and conventional optical microscopes
- Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue