Computational protocol: Diabetic pdx1-mutant zebrafish show conserved responses to nutrient overload and anti-glycemic treatment

Similar protocols

Protocol publication

[…] Larvae for live imaging were anesthetized using 0.003% Tricaine, immobilized in 1.5% low melt agarose and imaged on a Leica DM6000B microscope equipped with a SPOT-RT3 digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI), using a 20X water objective. Fluorescence stack and DIC images were captured using Visiview software (Visitron Systems, Puchheim, Germany). Fluorescence stacks were processed using ImageJ. Stacks were combined to a single image using an extended depth of focus projection. Adjustment of brightness and contrast, applied equally to all images, overexposed the signal of the islet to reveal the weaker signal present in single cells and small clusters located outside of the principal islet. Immunostainings of whole mounts were imaged with a Zeiss LSM5 Exciter confocal laser microscope using a 40X water immersion objective and a z-step ranging from 1–2μm. Stacks of optical sections were combined using a Maximum Intensity Projection (ImageJ). Insulin+, Glucagon+, Hormone+ and NeuroD:eGFP+ cells were quantitated using the PointPicker tool of ImageJ. Antibody signals were examined in combination with DAPI-staining of nuclei to facilitate cell identification. Confocal Images were processed with a median filter to remove speckle noise and assembled into composites using the FigureJ plugin of ImageJ and Adobe Illustrator. Insulin signal volume was quantitated using Imaris 7.3.0 (Bitplane). In brief, images were processed by smoothing (0.5 um) and local background subtraction, then a contour surface was created that enclosed the fluorescent signal, and the volume in voxels was determined. Signal threshold and object size filtering were applied consistently to all images. […]

Pipeline specifications

Software tools ImageJ, Adobe Illustrator, Imaris
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Danio rerio, Homo sapiens
Diseases Diabetes Mellitus, Hyperglycemia, Hyperglycemic Hyperosmolar Nonketotic Coma, Liver Diseases, Machado-Joseph Disease
Chemicals Glucose