Ix81 motorized inverted fluorescent microscope

Ten μm-thick coronal brain sections taken on a cryostat (CM3050; Leica; Buffalo Grove, IL, USA) were used for immunofluorescence analysis to examine galanin (1:200 dilution, T-4334; Peninsula Laboratories T-4334; San Carlos, CA, USA) immunoreactivity in the hippocampus. Tissues were washed with PBST (PBS + 0.1% Triton X100) prior to antigen retrieval with 10 mM sodium citrate (pH 6.0). Tissues were blocked in 3% PBST (PBS + 3% BSA + 0.4% Triton X100) for one hour before being incubated with the primary antibody overnight in a humidified chamber at 4°C. The next day tissues were washed with PBST then incubated with the secondary antibody (1:500, Alexa 488; Abcam; Cambridge, MA, USA) and DAPI (1 mg/ml diluted 1:500; Thermo Scientific; Waltham, MA, USA). Slides were washed again and coverslips were mounted with glycerol (G7893, 70% in water; Sigma-Aldrich; St. Louis, MO, USA). Images were captured on an Olympus IX81 Motorized Inverted Fluorescent Microscope (Center Valley, PA, USA) and quantified by calculating corrected total cell fluorescence [CTCF = Integrated density − (area of selected cell × mean fluorescence of background readings)] using Image J software (NIH; Bethesda, MD, USA).

Zebrafish larvae (Tg(fli1:EGFP)y1) at six-day post-fertilization (6-dpf) were exposed to 2-logs (from 5 to 100 µM) of the peptide. The mortality of zebrafish exposed to peptides was determined by observing the presence of heartbeat absence under a light microscope. Zebrafish larvae were separately exposed to a fixed concentration (20 µM) of the peptide for 3 h, then collected and mounted on microscope glass slides. An IX81 motorized inverted fluorescent microscope (Olympus Co., Tokyo, Japan) was used to monitor the biodistribution of the peptide in zebrafish.