Guidelines and Best Practices for O-GlcNAcylation, OGT, and OGA Imaging in Fixed Cells and Tissues

Abstract

O-GlcNAcylation is a nutrient-sensitive post-translational modification, dynamically cycled by the opposing enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). As its detection by any single reagent remains partial and uneven, the imaging literature on O-GlcNAc has yet to converge on a standardized approach to fixation chemistry, antibody choice, and image quantification, which challenges direct cross-study comparison. To navigate O-GlcNAc complexity for fixed-cell imaging, we provide an actionable, model-organism-specific methodological framework spanning sample fixation, permeabilization, blocking, detection chemistry, acquisition, and validation for the O-GlcNAc modification itself as well as for OGT and OGA. We further propose a minimal reporting checklist, aligned with current community standards for quantitative fluorescence microscopy, intended to let readers judge, reproduce, and build on O-GlcNAc imaging data.