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Expanding the toolset of fluorescent covalent staining of biological samples by labeling carboxylate and phosphate groups

Kexin Hu,Qimeng Sun,Ruifen Chen,Tinghao Xu,Yuncheng Li,Lili Chen,Aidong Wang,Hejing Qi,Danni Shao,Huanning Yue,Yaning Wang,Ziqi Tang,Yi Wang,Chunfeng Liu,Haijun Lv,Fen Wang,Huizhong Xu

Recently, fluorescent covalent staining methods have been developed for visualization of anatomical structures in cells and tissues. Coupled with expansion microscopy, these stains revealed various ultrastructural details. However, the covalently stainable chemical groups have been limited to amines, carbohydrates, and thiols. Here, we developed procedures for covalently labeling tissues for carboxylate and phosphate groups, utilizing carbodiimide crosslinker chemistry. In porcine kidney tissues, the carboxylate and phosphate stain provides 1.8–4.8-fold higher signal intensity than those from the three existing stains. In cancer cells, such stain allows 2–8-fold more accurate identification of nucleoli than the amine stain. In expansion microscopy samples, such stain reveals a variety of sub-cellular structures in tissues when combined with the amine stain. Such stain also allows imaging of lipid-based structures in cultured cells. With these advantages, this new covalent staining method further expands the toolset for fluorescent visualization of histology.