Zen black software

Colorimetric whole-mount in situ hybridization samples and live worm or fragment images were acquired using a Leica M205 microscope using the Leica Application Suite (LASX). Following image acquisition, non-tissue background was subtracted, contract and image intensity adjusted, and the edited image was converted to grayscale for data presentation. No quantifications were performed on contrast or intensity adjusted images and all raw, original data is available in the original data repository (http://www.stowers.org/research/publications/libpb-1513). Confocal images of fluorescent in situ hybridization samples were acquired using an LSM-700 inverted confocal microscope with Zeiss Zen Black Software (v8.1) or via high throughput imaging on a Nikon Eclipse Ti with a Yokogawa W1 spinning disk and robotic plate loader. With automated image capture, whole slides were imaged at 4X and objects of interest were automatically detected, re-imaged at 10X, then batch stitched, quantified and aligned using Fiji macros (https://github.com/jouyun/smc-macros). Stitching of tiles for whole worm images was performed with Fiji plugins (grid/collection stitching) using custom macros for batch processing. Maximum intensity projections of the stitched z-stacks were generated to visualize gene expression across the entire animal, then rotated, and cropped for data presentation.