Spectral imaging confocal microscope digital eclipse c1si

Mice were anesthetized with 1% sodium pentobarbital and transcardially perfused with normal saline followed by 4% PFA in 0.01 M PBS. Mouse brain was dissected out and postfixed with 4% PFA/PBS for 4 h at 4°C. Fixed brain was incubated with 20% sucrose overnight and then 30% sucrose overnight. The brain was embedded in optimal cutting temperature compound and stored at −80°C until usage. 30-μm cryosections were made using cryostat and collected.
For immunostaining of brain sections, floating 30-µm-thick slices were rinsed with PBS and permeabilized in 0.4% Triton-X 100 in 0.01 M PBS for 30 min. Cryosections were blocked with 1% BSA in PBS containing 0.4% Triton-X 100 for 1 h at RT and then incubated with primary antibodies overnight at 4°C. Secondary antibodies conjugated with Alexa Fluor 555 were used for detection. Sections were then incubated with DAPI (Roche) for nuclear staining for 5 min at RT. Following rinsing, cryosections were mounted on gelatin-coated slides and covered with coverslip with mounting medium. Confocal images were collected using the Spectral Imaging Confocal Microscope Digital Eclipse C1Si (Nikon) with a 10× Plan Apochromat differential interference contrast N1 0.45 objective or 40× Plan Fluo (NA 1.30) oil objective (Yang et al., 2018 (link)).

Neuro-2a cells expressing EGFP-EEN1 were incubated with modified KRH buffer and imaged live at 37°C using a 100× Plan Apochromat VC (NA 1.40) oil objective with the Spectral Imaging Confocal Microscope DIGITAL ECLIPSE C1Si (Nikon). Cells untreated or treated with W-7 (20 µM) for 20 min were imaged every minute for two frames, supplemented with ionomycin (1 µM), and imaged every 10 s to 1 min for 30 min.