H_CA undergoes axonal retrograde transport with the neurotrophin receptor p75^NTR in microfluidic chambers.

<p>(<b>A</b>) Axonal compartment of a MFC. Motor neurons differentiated from an embryonic stem (ES) cell line expressing GFP under the control of the motor neuron-specific promoter (HB9::GFP) have been seeded in the somatic compartment (on the left; not shown). Upon differentiation, their axons elongated through the microgrooves (arrow in the <i>merge</i> panel) and reached the axonal compartment (hatched lines indicate the boundary of this compartment). Cells were stained for the neuronal marker βIII tubulin (blue) and the axonal marker SMI32 (red). Cell bodies are out of view on the left. Scale bar, 50 µm. (<b>B</b>) Primary mouse motor neuron cultures in MFC were used to assess the axonal retrograde transport of H_CA. Motor neurons were incubated with fluorescent H_CA (red, on the left) and with a fluorescent antibody for p75^NTR (green, middle), for 30 min at 37°C in resting conditions, then washed and imaged. Both H_CA and the antibody have been added to the axonal side only of the MFC. Representative stills from a confocal time series are shown in the top part of the panel whilst the relative kymographs are shown at the bottom. Arrowheads indicate double-positive retrogradely transported organelles. Cell bodies are located out of view on the right. The analysis shown in (<b>B</b>) is representative of two independent primary motor neuron cultures. Scale bar, 20 µm.