Live-cell imaging of MV151-labeled proteasome subunits

Fluorescent labeling of proteasomes was performed as described (Verdoes et al., 2006). For live-cell imaging of proteasome transport across the AIS region, we preincubated neurons with pan-neurofascin antibody (A12/18) for 30 min to label the neurofascin extracellular domain that enabled us to detect the AIS, followed by 1-h incubation with proteasome-labeling agent MV151 (100 µM) and anti-mouse secondary antibody Alexa Fluor 488 conjugate (ab150113, Abcam) at 37°C in culture medium. Neurons were then visualized with a 6× oil-immersion objective (NA 1.4; Zeiss) in extracellular buffer (145 mM NaCl, 10 mM Hepes, 8 mM glucose, 3 mM CaCl₂, 2 mM MgCl₂, and 3 mM KCl) on a Zeiss Observer.Z1 microscope integrated with an AxioCam MRm camera and temperature/CO₂ modules. Zen blue software (Zeiss) was used for microscope control and data acquisition (0.5 Hz for 3 min, 90 frames total). MV151 was excited at λ_ex = 543 nm and detected at 560–620 nm.