Red fluorescent MDW941 (5 or 100 nM) and green fluorescent MDW933 (500 nM) were used to label active GCase (Witte et al., 2010 (link)) and were a gift from Dr. E. Sidransky (NIH/NHGRI) (Westbroek et al., 2016 (link)). BODIPY-pepstatin-FL (1 μM, Thermo Fisher Scientific) was used to label active cathepsin D. For lysosome anterograde delivery assays, 100 μL of NFM containing fluorescent probe was applied on the soma chamber, while applying 200 μL NFM without probe on the axon chamber to maintain fluidic separation between soma and axon chambers. For fixed-cell imaging, live neurons were incubated with MDW933 (500 nM) applied on both soma and axon compartments for 1 hour to label all active GCase containing lysosomes, or with MDW933 (500 nM) applied only on the soma compartment for 15 min followed by varying washing times in NFM as indicated to label active GCase containing lysosomes delivered to distal axons. MDW933 labeled neurons were fixed and immunolabeled for β3-Tubulin as described above. For live imaging, neurons were incubated in NFM containing the various probes for 30 min at 37°C, followed by 3 washes with NFM.
For labeling lysosomal cargo degradation in axons, Magic Red Cathepsin B/L fluorogenic substrates (1:4,000, ImmunoChemistry Technologies) were applied to the axonal chamber for 30 min at 37°C to monitor Cathepsin B/L-mediated degradation.
For labeling lysosomal cargo degradation in axons, Magic Red Cathepsin B/L fluorogenic substrates (1:4,000, ImmunoChemistry Technologies) were applied to the axonal chamber for 30 min at 37°C to monitor Cathepsin B/L-mediated degradation.