Dm6 confocal microscope

A BODIPY/egg yolk mixture was prepared by emulsifying sterile 5% chicken egg yolk in zebrafish embryo media via sonication for 3–5 min (Carten et al., 2011 (link)). BODIPY dissolved in DMSO (Thermo Fisher, Waltham, MA) was quickly added to the emulsified egg mixture at a final concentration of 6.4 μM and vigorously vortexed for 3–5 min. 1ml of the BODIPY/egg yolk mixture was added to 4dpf zebrafish in a sterile 12-well plate (10 zebrafish/well) for 5 hrs. Zebrafish were covered in foil during their exposure to the BODIPY/egg yolk mixture. To visualize the patency of the pancreatic ductal system, the confocal feature of a LEICA DM6 confocal microscope (Leica Microsystems Inc., Buffalo Grove, IL) was used for imaging live zebrafish administered BODIPY/egg yolk. With DIC, the pancreas and islet were located and z-stack images throughout the entire head of the pancreas were acquired. The presence or absence of BODIPY within the pancreas was assessed by compiling the z-stacks taken of the pancreas into maximum projections.

For oral microgavage, larval zebrafish were anesthetized, mounted in sterile 4% methylcellulose and gavaged as described previously (Cocchiaro and Rawls, 2013 ). Proteins were administered to each larval zebrafish with a 4.6 nL volume at an injection rate of 23 nL/sec using a Nanoject II Auto-Nanoliter Injector (Drummond Scientific Company, Broomall, PA). To visualize the global localization of BefA in vivo, mCh-BefA, mCh, mNG-BefA, or mNG were administered at a concentration of 1 mg/mL. All images were taken no longer than 2 hrs after gavage using the wide-field feature of a LEICA DM6 confocal microscope (Leica Microsystems Inc., Buffalo Grove, IL).
For cardiac valley injections, 4 dpf larval zebrafish were anesthetized and injected directly into the cardiac valley with 2 pg of BefA or negative control vehicle again using the Nanoject II Auto-Nanoliter Injector (Drummond Scientific Company, Broomall, PA) as previously described (Wiles et al., 2009 (link)). Zebrafish were revived and housed in separate wells of a 24-well plate after the injection to monitor health. To ensure that gavaged or injected GF larvae were not contaminated with microbes during these procedures, a sample of embryo media and homogenized gastrointestinal tracts from some larvae were plated onto tryptic soy agar (TSA) or luria broth (LB) agar at the time of harvest, typically 48 hrs later.