SidK-AL568 detects the V-ATPase in acidic intracellular organelles. (A) HeLa cells were stained with SidK-AL568 (magenta). Nuclei labeled with DAPI (blue). Arrowheads mark peripheral, likely endocytic, vesicles (open) and juxtanuclear vesicles and cisternae, likely including the Golgi compartment (closed). (B–F) V-ATPase in HeLa cells labeled with SidK-AL568 (magenta) and costained for (B) LAMP1, (C) trans-Golgi, (D) cis-Golgi, (E) ER, and (F) mitochondria (green). Nuclei labeled with DAPI (blue). Side panels show individual SidK-AL568 and organelle channels in area denoted by dashed square, at 1.1, 0.9, 1.1, 1.7, and 1.5× magnification, respectively. (G and H) HeLa cells were immunostained using the V₁A antibody, α-ATP6V1A (Abnova; catalog no. H00000523-M02; magenta) in cells costained for LAMP1 (G) or trans-Golgi (H), in green. Nuclei labeled with DAPI (blue). Side panels show individual α-V₁A and organelle channels in dotted square, at 1.1 and 1.4× magnification, respectively. (A–H) are extended focus images representative of ≥30 fields from three or more experiments. Colocalization and Mander’s coefficients (M) between the organellar marker and V-ATPase probe shown in orange. All scale bars: 5 µm.