Lumar v12 fluorescence stereomicroscope

Use of the chick CAM to model the formation of metastatic colonies and primary tumours is described in detail elsewhere17 (link)18 (link). In brief, fertilized eggs decanted into sterilized weigh boats were allowed to mature ex ovo for 9 days. GFP-expressing cells were injected either i.v. or within the CAM tissue on developmental day 10. At indicated times, the ex ovo chick embryos were placed in a custom intravital imaging chamber46 (link)47 (link). Images of tumour cells in the CAM were acquired using a Lumar V12 fluorescence stereomicroscope (Zeiss) or an Olympus BX61 equipped with a digital camera controlled with Volocity image acquisition software (PerkinElmer).

Well-fed synchronized animals were picked onto new 90 mm NGM plates seeded with a uniform lawn of OP50E. coli. These plates were seeded 12 hr before the start of the assay and the bacteria were allowed to grow at 20°. The animals were allowed to equilibrate for 1 min after transfer to the assay plate. For all analysis, a 3-min video was made using a Zeiss Lumar V12 fluorescence Stereomicroscope. This video was then segmented into shorter videos of 20 sec using custom code written in MATLAB (MATLAB Release 2012b; The MathWorks, Natick, MA). The 20-sec-long video was then scored by eye for body bends. A body bend was defined as a change in the direction of propagation of the part of the animal corresponding to the posterior bulb of the pharynx along the y-axis, assuming the C. elegans was traveling along the x-axis (Tsalik and Hobert 2003 (link)).