Multiplexing Carbotrace and Antibodies

Multiplexing Carbotrace and Antibodies

When trying to understand and build in a world reliant on polymers, plant tissue is at the forefront of green chemistry with the abundance provided by polysaccharides. Techniques for the analysis of structures, morphology and localisation of different carbohydrates provides deep insight in a variety of fields, making in depth imaging and visualising crucial to basic research.

When working with varied polysaccharides or other structural elements like proteins, researchers need to be able to distinguish components in complex environments such as within cells or in multicellular organisms reliably, which requires specificity.

Currently one of the most commonly used techniques are antibodies. Antibodies are proteins employed by the immune system of animals to bind and mark antigens which are alien to the host's body such as viruses or foreign bacteria. These proteins can be functionalised by the binding of a fluorescent marker. Antibodies however tend to be large and require a specific target to be effective, working best with proteins leaving polysaccharides largely under tagged.

Addressing this gap, Carbotrace can be used to target the native glycosidic linkages found in unbranched or moderately branched polysaccharides. Carbotrace are small structure-responsive fluorogenic molecules, highly fluorescent when binding and presenting a unique spectral fingerprint depending on polysaccharide configuration. This manifests in blue or redshift of the fluorescent spectrum. Using this technique several glucans can be imaged and identified in parallel.

A strong advantage is the seamless way that antibodies and Carbotrace can be paired or multiplexed when used on the same tissues. This provides in-depth localisation across a variety of polysaccharides and proteins. This versatility is possible by pairing different fluorescent channels, allowing further analysis with established methods with little disruption.

To exemplify this, Plachno and colleagues from the Jagellonian University, in Poland, investigated gland cell walls in Venus flytraps by multiplexing protein specific cell wall antibodies and Carbotrace 680 (see image). The researchers found specialised arabinogalactans and hemicellulose distributions within delicate in-grown regions that identified modulable composition in cell walls. In conclusion, multiplexing Carbotrace and Antibodies provides a simple method to build a deeper understanding into plant tissues by simultaneously exploring carbohydrates and proteins.

Image: Basal cell (Bc), stalk cell (Sc), secretory cells (white star) and  glandular cells (green star) were all stained with Carbotrace 680 (in red) and LM15 antibody (in green) to identify cell wall ingrown composition. Scale bar: 10 µm. Image adapted from Figure 6B by Płachno, B. et al. (2026) International Journal of Molecular Sciences, 25(23), 13124. (CC BY 4.0).

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