Endosomal Transport to Lysosomes and the Trans-Golgi Network in Neurons and Other Cells: Visualizing Maturational Flux.

High-level microscopy enables the comprehensive study of dynamic intracellular processes. Here we describe a toolkit of combinatorial approaches for fixed cell imaging and live cell imaging to investigate the interactions along the trans-Golgi network (TGN)-endosome-lysosome transport axis, which underlie the maturation of endosomal compartments and degradative flux. For fixed cell approaches, we specifically highlight how choices of permeabilization conditions, antibody selection, and antibody multiplexing affect interpretation of results. For live cell approaches, we emphasize the use of sensors that read out pH and degradative capacity in combination with endosomal identity for elucidating dynamic compartment changes.