Distinguish surface and intracellular signal
Compare matched time points and inspect cell edges separately from intracellular regions. Intracellular signal in live cells may reflect uptake; a single endpoint image cannot establish its origin or trafficking route.
Check localization evidence
For organelle localization, use an appropriate independent marker and single-stain controls to exclude spillover. Keep exposure and display ranges consistent so automatic contrast changes are not mistaken for redistribution.
Plan surface and intracellular comparisons
Quantify cell-edge and intracellular regions separately and record intervals between staining, washing and acquisition. Before using fixed samples or low-temperature comparisons, assess effects on cell state and glycan accessibility so treatment effects are not mistaken for uptake.