Spinning disc laser confocal microscope

E12.0–E12.5 urogenital ridges were dissected in cold PBS and then cultured in organ culture medium consisting of Dulbecco’s modified Eagle’s medium (with 4.5 g/ L D-glucose, without sodium pyruvate and phenol red), 10% fetal bovine serum, 2 mM glutamine and 1 mM sodium pyruvate (Invitrogen Inc.). A culture system for fetal gonads and urogenital tissues was described previously (Nel-Themaat et al., 2009 (link)). Briefly, urogenital ridges were placed on a Millicell tissue culture plate insert (Millipore Corporation) and cultured at the air-medium interface in a 35 mm glass bottom culture dish (MatTek Corporation). Time-lapse images were acquired on a PerkinElmer spinning disc laser confocal microscope at 37°C and 5% CO₂ in a humidified chamber. A 514 nm laser was used to acquire YFP images. Images were taken at 100x or 200x magnifications (with 10x and 20x long working-distance objective lens) using 50% laser power and 300 millisecond exposure with 2 × 2 binning. The Z-plane depth was 40–60 μm. Images were processed by Ultra VIEW ERS ImageSuite Software (PerkinElmer Life and Analytical Sciences, Inc.) and Adobe Photoshop.