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Pipeline publication

[…] induction, embryos were incubated at 39°C for 2 h and fixed 90 days post induction (dpi). After 1, 10, 30 or 150 days, fish were sacrificed, fixed in 4%PFA/2× PBST overnight at 6°C, and divided into two groups. One group was cryopreserved in 30% sucrose, embedded in Leica tissue freezing medium and sectioned at 30-50 µm on a cryostat. Confocal images of the sections were collected using a Leica SPE and processed using the Leica application suite X software (LASX) and Adobe Photoshop CS4. From the other group, guts were dissected and used for whole gut imaging with a Nikon SMZ18 microscope and Luxendo GmbH (Heidelberg) 25× MuVi-SPIM. In general, image processing was conducted with Fiji (; ). Imaris software was used for 3D reconstructions. Surfaces of clones and the related movies were rendered with Chimera from UCSF (). Raw data movies of GaudíLxBBW were rendered with Vaa3d (). Quantification of clone size was conducted by thresholding for nuclei and measuring clone volume with the BoneJ Command ‘Particle Analyser’ (). This clone size was divided by a standard size of cell nuclei found in the data (4.4 µm³) and rounded down to next full number. Subsequently, clones consisting of no cells were discarded as artefacts. GraphPad Prism v.7 (www.graphpad.com) was used for statistical analysis and visualization of the data., Immunostaining and Haematoxylin and Eosin (H&E) staining were performed as described previously (), using the following primary antibodies (all at 1:500): chicken anti-EGFP (Life Technologies, A10262), rabbit anti-phospho H3 (Upstate Biotechnology, 06-570) and rabbit anti-caspase 3 (Abcam, ab13847). Nuclear DNA was stained with DAPI (Sigma, D9564) or […]

Pipeline specifications

Software tools Imaris, Vaa3D, BoneJ