A1 si laser scanning confocal microscopy

For direct assessment of β-cell proliferation, the Click-iT EdU Alexa Fluor 647 Imaging kit (Life Technologies) was used. For larval studies, drug treated 3 dpf larval were treated with EdU (working concentration 12.5 µM) and fixed at 5 dpf. In adult fish, 25 μM EdU in E3 medium was injected intracoelomically every other day together with paroxetine or 0.1% DMSO control for 10 days and alone on the twelfth day. In both cases, pancreata were dissected out, embedded in paraffin blocks, sectioned and stained as per the manufacturer's instructions. Images were collected with a Nikon A1-si Laser Scanning Confocal microscopy. EdU-positive β cells were counted using the ImageJ (NIH) software. All p-values were calculated using Student's t-test as comparisons were made only between a single treatment condition and the control.

Fish were fixed either in 4% PFA (5 dpf larvae) or 10% formalin (adults) at 4°C overnight. Pancreata were dissected and immunohistochemistry performed as described previously (Huang et al., 2014 (link)). Briefly, pancreata were embedded in paraffin and sectioned at 5 μm. Sections were stained with 4', 6-diamidino-2-phenylindole (DAPI) and processed for immunostaining using the following primary antibodies: serotonin (5-HT; 1:100, Rabbit polyclonal, ImmunoStar); acetylated tubulin (aTub; 1:400, Mouse monoclonal, Sigma); green fluorescent protein (GFP; 1:400, Rabbit polyclonal, Life Technologies), GFP (1:400, Mouse monoclonal, Life Technologies), DsRed (1:400, Mouse monoclonal, Clontech); insulin (1:400, Polyclonal Guinea Pig, Dako). Fluorescently conjugated secondary antibodies were diluted 1:400 dilution (Jackson ImmunoResearch Labs). Images were collected using a Nikon A1-si Laser Scanning Confocal microscopy.

To validate Hit I and II compound effects in promoting increased β-cell numbers, the ins:hmgb1-EGFP (Parsons et al., 2009 (link)) line was used; this line facilitates β-cell quantification due to nuclear localization of the GFP reporter. The assay was performed similarly to validation I tests except that Hit drugs were tested from 25 μM to 0.2 μM using a 1:5 dilution series. Pancreata were dissected and imaged with a Nikon A1-si Laser Scanning Confocal microscopy under a 20× objective. β cells were counted for all Z planes using ImageJ (NIH) software. All assays were evaluated using one-way ANOVA (Analysis of Variance) and p-values calculated with a post hoc Dunnett's test. n = 5–10 larvae per condition, and a minimum of three experimental repeats was performed.