Dragonfly spinning disk system

4–5 days after transfection with either PPO-Venus or MOR-GFP, neurons were transferred to a 37°C heated chamber filled with extracellular solution containing (in mM): 145 NaCl, 3 KCl, 2 CaCl₂, 1 MgCl₂, 10 glucose, 10 HEPES, pH to 7.3 with NaOH, 310 mOsm. To avoid prior photoactivation, transfected cells were first identified using low light then coverslips were pretreated with 10 μM 9-cis-retinal for at least 30 minutes in the dark prior to imaging. Live-cell imaging was performed on an Andor Dragonfly spinning disk system described above using the 488 nm laser line for Venus/GFP Z-stack imaging and a sCMOS camera (Andor Zyla) acquired every 5 minutes. Neurons were photoactivated with either a blue LED at 10 Hz described above for TIRF imaging or treated with 1 μM DAMGO, and imaged for 30 minutes.

All procedures were performed as described (Anczuków et al., 2012 (link)). Microscopy was performed on a Zeiss Axiovert 200M instrument using the ApoTome imaging system (Zeiss) or the Dragonfly Spinning Disk system (Andor). T7 (CSHL antibody facility), cleaved caspase-3 (Cell Signaling) and ki67 (Zymed) primary antibodies were used at 1/50, 1/100 and 1/100 dilutions, respectively. Alexa Fluor 568 anti-mouse and 488 anti-rabbit secondary antibodies (Invitrogen) were used at 1/500 dilution. Acini were scored positive for ki67 when at least five cells within the acini were stained and positive for cleaved caspase-3 when at least one cell in the lumen was stained.