Is the direct approach the better choice in high-plex Spatial Biology?
von Leo Schroeder
Beyond legacy staining:
While secondary antibodies served the industry well for simple immunohistochemistry, they have become a primary bottleneck in the era of high-plex spatial biology. "Legacy" staining methods are simply not designed for the complexity of e.g. modern tumor microenvironment analysis. The reliance on secondary antibodies introduces bottlenecks that compromise reproducibility, sensitivity, and time-to-result. To achieve the robustness required for modern spatial biology, we must move beyond these limitations.
Challenges and Constrains in High-Plex Imaging
Using secondary anti-species antibodies in spatial multiplex assays presents numerous challenges that can compromise both experimental timing and the quality of results, often requiring complex optimizations and careful methodological choices resulting in a panel design bottleneck.
One of the most pervasive problems is cross-reactivity between secondary antibodies and endogenous immunoglobulins found in tissues, such as human, mouse, or rat samples. This interaction can lead to high background staining and false positives that obscure true biological signals, especially in regions rich in Fc receptors like the spleen and lymph nodes or among macrophages and microglia. Mouse-on-mouse and rat-on-rat staining are particularly problematic, driving up the need for extensive blocking steps that consume critical experimental time and sometimes fail to yield consistently clean results.
A related challenge is the necessary reliance on species-specific secondary antibodies which severely restricts panel design. Because the majority of high-quality monoclonals are mouse- or rabbit-derived, mixing targets from the same species inevitably causes cross-reactivity and signal bleed-through. This lack of flexibility limits panel size and forces researchers into a difficult choice: Accept lower-plex data or invest in expensive custom antibodies and time-consuming optimization steps.
Iterative multiplex protocols such as tyramide signal amplification or cyclic immunofluorescence pose significant risks to data integrity. The harsh buffers and heat required for antibody stripping compromise the tissue epitopes, leading to signal loss or misinterpretation in later rounds. Furthermore, the added bulk of secondary antibodies creates steric hindrance, limiting tissue penetration in dense structures like nerve bundles or tumor cores. This physical constraint reduces staining intensity and increases non-specific binding, ultimately compromising assay sensitivity.
Figure 2: High-plex spatial imaging examples obtained with the RareCyte Orion platform
Non-specific background is amplified by secondary antibodies binding to Fc receptors, sticky extracellular matrix regions, or highly autofluorescent tissue domains like those containing lipofuscin, collagen, or elastin. While blocking strategies are used, their efficacy is not always sufficient, particularly in complex tissues, which again necessitates extra time spent in protocol adjustments and repeated controls. Additional complications arise when limited species diversity makes it difficult to assemble large multiplex panels. Most commercial antibodies are either mouse or rabbit, with others (goat, donkey, chicken) offering only partial coverage, rendering high-plex spatial assays labor-intensive and restrictive.
Registration and signal alignment issues combined with fluorescence carryover between cycles can lead to pixel-shift artifacts, misrepresenting co-localization data. The success of multiplex imaging relies heavily on uniform staining, which is affected by variables such as tissue fixation (FFPE versus fresh frozen), antigen retrieval protocols, and intrinsic autofluorescence, all requiring time-consuming sample-by-sample optimization to prevent misleading artifacts.
Finally, the expansion of more channels in fluorescence-based methods increases the risk of spectral overlap and bleed-through, with broad-spectrum fluorophores and autofluorescent tissues compounding these issues. Researchers attempting to push the boundaries of multiplexing often favor directly conjugated primary antibodies, DNA-barcoded primaries, or advanced tyramide signal amplification systems that leverage covalent fluorophore attachment and more sophisticated blocking to bypass secondary antibody pitfalls.
Figure 3: High-plex spatial imaging examples obtained with the RareCyte Orion platform
Moving Toward More Scalable and Reliable Multiplexing
Recent reviews and technology guidelines consistently emphasize the need for robust workflows, rigorous antibody validation, and the implementation of newer multiplexing solutions to maximize signal fidelity while reducing turnaround time and error rates. Where possible, alternatives such as Rarecyte´s Orion system facilitate broader panels and minimize cross-reactivity, enabling high-quality spatial data acquisition at scale.
For further reading, consult e.g. Semba et al. (2024, Nature Cancer), Carstens et al. (2024, PMC), Hickey JW et al. (2021, PMC), and relevant technical guidelines from e.g. Cell Signaling.