Microscopy and image processing

Routine bright-field and fluorescence imaging of zebrafish used an Olympus MVX10 stereo dissecting microscope (Olympus, Tokyo, Japan) with MV PLAPO 1× and 2×C objectives, fitted with an Olympus DP72 camera and Cellsense standard software, version 1.11.

Confocal intravital microscopy used a Zeiss LSM 5 Live with a Plan-Apochromat 20×, 0.8 NA objective (Zeiss, Oberkochen, Germany). ZEN software (2012, black edition, 64-bit) was used for acquisition, and images were 16-bit 512 × 512 pixels. Z-depth ranged from 35–130 μm (72 ± 23 μm) and was composed of 20–40 slices (31 ± 4). Time intervals between z-stacks were set as zero to perform continuous acquisition (z-stack acquisition took 33.24 ± 9.50 s). Excitatory laser wavelengths were 405 nm for calcofluor, 489 nm for EGFP, and 561 nm for mCherry. Emission detection used a BP495-555 filter for calcofluor and EGFP and an LP575 filter for mCherry. Excitation/emission conditions for light yellow particles were the same as for calcofluor.

Details of microscopes, cameras, and acquisition software used for other experiments are provided with their respective methods.