Fluorescence Imaging of Ventral Nerve Cord

Fluorescence imaging was performed as previously described (Kowalski, Dahlberg & Juo, 2011 (link)). Briefly, nuIs24 or nuIs24; dmd-10(gk1131) young adults were immobilized using 30 mg/ml 2,3-butanedione monoxamine (Sigma-Aldrich, St. Louis, MO, USA) and the anterior ventral nerve cord immediately posterior to RIG was imaged (Juo et al., 2007 (link)). A total of 1 μm (total depth) Z-series stacks were collected using a Carl Zeiss Axiovert M1 microscope with a 100X Plan Apochromat (1.4 numerical aperture) objective equipped a GFP filter cube. Images were collected with an Orca-ER CCD camera (Hamamatsu) and MetaMorph (version 7.1) software (Molecular Devices). Maximum intensity projections of Z-series stacks were used for quantitative analyses of fluorescent puncta. Exposure settings and gain were adjusted to fill the 12-bit dynamic range without saturation and were identical for all images taken. Line scans of ventral cord puncta were generated using Meta-Morph (version 6.0) and were analyzed using custom written software (Burbea et al., 2002 (link)) with Igor Pro (version 6 Wavemetrics). Statistical analyses were performed using Student’s t test.