Alexa 488 conjugated dextran

After retrograde identification of M1 and the frontal premotor cortex (PM) as the areas that originate porcine corticospinal axons, anterograde neural tracers were injected in those areas in three additional animals. For this, a craniotomy was performed on the left side of the skull over the cruciate and the superior frontal gyri, removing the bone from the midline to about 3 cm lateral, and from 3.5 cm rostral to 0.5 cm caudal to the frontal-parietal suture. Two different tracers were injected to study the terminations of axons arising from each cortical region. Alexa 594-conjugated dextran (10,000 MW, Thermo Fisher Scientific Inc., D22913) was injected in M1, and Alexa 488-conjugated dextran (10,000 MW, Thermo Fisher Scientific Inc., D22910) was injected in PM. Both tracers were dissolved at 10% in saline solution. Twelve injections (4-μL each, at 2 mm and 4 mm of depth in the dorsoventral plane, separated 0.8-mm along the longitudinal axis of the respective cortical gyrus) of each tracer were made using a 50-μL Hamilton syringe mounted in a micromanipulator. The needle was set in place for 4 min to prevent reflux of the injected solution. The same total dose of 48 μL of each tracer was administered in all animals. The dura matter and scalp were closed separately.

Lysosomes were labelled by incubating cells with 200 μg/mL Alexa⁵⁴⁶-conjugated dextran or with 200 μg/mL Alexa⁴⁸⁸-conjugated dextran (Thermo Fisher Scientific, Mississauga, ON) or with 2.5 mg/mL Lucifer yellow (Thermo Fisher Scientific, Mississauga, ON) for 2 h in complete media at 37°C in 5% CO₂. Cells washed with phosphate-buffered saline (PBS) and resupplied with complete cell-specific media for 1 h to chase the fluid-phase marker to lysosomes before pharmacological manipulation and live-cell imaging. We note that we use “lysosomes” to represent a potential mixture of late endosomes, lysosomes and endolysosomes [5 (link), 30 (link)]. Lysosomal calcium was labelled with Fluo-4AM 8 μM by pulsing for 45 min in complete media at 37°C in 5% CO₂, followed by washing with PBS and addition of complete media for 45 min to chase the marker to lysosomes.