Anti fading fluorescent mounting medium

Cytospins were made directly from FACS-sorted cells on non-coated slides. Cells were fixed in ice-cold 100% methanol for 15 min at -20°C, rinsed three times in phosphate buffered saline (PBS), and then blocked in Blocking Buffer (1XPBS/5% normal serum/0.3% Triton™ X-100) for 60 min. Indirect immunostaining was performed at 4°C overnight using the polyclonal anti-LC3 primary antibody (1:200, Cell Signaling Technology, Inc. MA, USA) and rinsed three times in PBS. Then specimens were incubated in anti-rabbit IgG-Cy3 secondary antibody (1:200, Sigma-Aldrich, Saint Louis, USA) for 2 h at room temperature in the dark. Slides were then stained with DAPI to label nuclei. The anti-fading fluorescent mounting medium (Vectorshield, Vector Laboratories, Inc. Burlingame, USA) was added, and the cells were covered with coverslips. The slides were then imaged by confocal laser scanning microscopy (PerkinElmer UltraVIEW Vox, Cambridge, UK). Volocity software 4.0 was used to identify and quantify fluorescence intensity.

To verify viral expression, mice were deeply anaesthetized with isoflurane and transcardially perfused with PBS, followed by fixation in cold 4% PFA solution for 48 h. Then the brain tissue was dissected and fixed in PFA overnight at 4 °C. The brain tissue was dehydrated in PBS solution containing 30% sucrose and then embedded in OCT freezing embedding solution. Coronal sections (30 μm) were cut on a Leica CM1860 UV freezing microtome (20 sections per brain). The brain slices were permeabilized in PBST for 20 min, subsequently blocked with 5% BSA in PBST for 1 h at room temperature, and then incubated with anti-mCherry (Thermo Fisher M11217,1:1000) and anti-Serotonin (Sigma–Aldrich s5545,1:2000) overnight at 4 °C. After washing with PBS for 10 min and PBST twice for 10 min each, brain slices were incubated in Alexa Fluor 568 Goat anti-Rat (Thermo Fisher, 1:400) and Alexa Fluor 488 Donkey anti-Rabbit (Jackson Labs, 1:200) for 3 h at room temperature. After washing 3 times with PBS for 10 min each, brain slices were sealed with anti-fading fluorescent mounting medium (Vectorlabs) overlay and subsequently imaged with a confocal microscope.

A 4-week-old zebrafish was placed in chilled extracellular fluid and anesthetized. The brain tissue was dissected out and fixed in 4% PFA for 24 h. Then the tissue was washed three times each for 5 min with PBS and permeabilized in PBS containing 1% Triton X-100 (PBST) for 2 h. After 1 h of blocking with 5% BSA in PBST, the brain tissue was then incubated in anti-serotonin (Sigma–Aldrich S5545, 1:3000) for 48 h at 4 °C. After washing the primary antibody with PBS, the tissue was incubated with secondary antibody Alexa Fluor 488 Donkey anti-Rabbit (Jackson Labs 711-545-152, 1:200) overnight at 4 °C. After washing the secondary antibody with PBS, the brain tissue was placed on a slide with the dorsal side up and mounted with the anti-fading fluorescent mounting medium (Vectorlabs) for imaging. An Olympus FV3000 laser scanning confocal microscope was used for image acquisition.