Sensitive detection of lysosomal membrane permeabilization using the galectin puncta assay

The Lysosomal Galectin Puncta Assay is a microscopy-based technique able to detect even minor lysosomal leakage with high sensitivity. This protocol describes the detection of galectin puncta as markers of lysosomal membrane permeabilization, a process that relies on the high-affinity binding of the cytosolic galectins to the luminal glycans exposed on damaged lysosomes. Compared to traditional methods, the Galectin Puncta Assay offers high sensitivity, detects subtle lysosomal leakage, and enables analysis at single-lysosome level. Here, we provide a step-by-step protocol for this assay, covering sample preparation, immunostaining, imaging and image quantification.