Glycan Painting: Triplex Lectin Staining Enables Visualization of Cell-Type-Specific Glycan Profiles in Tissue Sections

Abstract Multiplex staining is a technique that allows the identification of cell types within a single tissue section by simultaneously detecting multiple molecular markers. Generally, multiplex staining is performed using several combinations of probes, including specific antibodies, nucleic acid probes, and lectins. Here, a novel multiplex staining strategy that relies exclusively on lectin probes that target glycans is presented. Glycans have a vast variety of structural forms that vary depending on cell type-specific modifications. Furthermore, an enormous number of glycan-binding molecules, collectively known as lectins, exist in the biological world. Each lectin displays specificity for a particular glycan motif while maintaining broad affinity. Although lectin-based cell staining has been used in various applications, the partial and limited specificity of lectins has hindered the use of glycan-targeted multiplex staining with lectins. In addition, lectin probes have largely been avoided for cell-type identification because of the absence of strict cell-type-specific glycans. Here, a novel staining method, Glycan Painting, is introduced. Rather than viewing the partial specificity of lectins and the broad, non-cell-type-specific distribution of glycans as drawbacks, this approach turns these features into advantages by generating distinct color patterns that comprehensively visualize cell-type-specific glycan combinations and enable full-color imaging of tissues. Competing Interest Statement The authors have declared no competing interest. Abbreviations - ConA - concanavalin A - H&E - hematoxylin and eosin - LEL - Lycopersicon esculentum (tomato) lectin - PBS - phosphate-buffered saline - WGA - wheat germ agglutinin - TBS - Tris-buffered saline.