Antibodies used were against GFP (1:500; Invitrogen, Life Technologies, Grand Island, NY, USA), quail endothelial marker Qh1 (1:10) and Pax7 (1:10) (both from Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA), desmin (1:100; MP Biomedicals, Santa Ana, California, USA) and SMA (1:200; Sigma-Aldrich, Rehovot, Israel), both recognizing smooth and striated muscle [65 (link)], and ZO-1 (1:100, Zymed, Life Technologies, Grand Island, NY, USA). Secondary antibodies were coupled either to Cy2 or Rhodamine (1:100; Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Nuclei were visualized with Hoechst.
Images were photographed using a DP70 (Olympus) cooled CCD digital camera mounted on a BX51 microscope (Olympus) [14 (link)]. Confocal sections of whole-mount preparations encompassing their entire thickness were photographed using a Nikon Eclipse 90i microscope with a Plan Apo 10x/0.45 dry objective (Nikon) and a D-Eclipse c1 confocal system (Nikon) at 2.7 μm increments through the z-axis. Images were z-stacked with EZ-C1 3.90 FreeViewer software. For figure preparation, images were exported into Photoshop CS3 (Adobe). If necessary, the levels of brightness and contrast were adjusted to the entire image and images were cropped without color correction adjustments or γ adjustments. Final figures were prepared using Photoshop CS2.
Images were photographed using a DP70 (Olympus) cooled CCD digital camera mounted on a BX51 microscope (Olympus) [14 (link)]. Confocal sections of whole-mount preparations encompassing their entire thickness were photographed using a Nikon Eclipse 90i microscope with a Plan Apo 10x/0.45 dry objective (Nikon) and a D-Eclipse c1 confocal system (Nikon) at 2.7 μm increments through the z-axis. Images were z-stacked with EZ-C1 3.90 FreeViewer software. For figure preparation, images were exported into Photoshop CS3 (Adobe). If necessary, the levels of brightness and contrast were adjusted to the entire image and images were cropped without color correction adjustments or γ adjustments. Final figures were prepared using Photoshop CS2.
