Movie 4.

Confocal z-stack using a 40x/1.25NA glycerol objective with 8x confocal zoom of animals immunostained to detect TUBA-2 (green) and FISH to detect collagen mRNA (magenta) and nuclei (blue). Movie progresses from internal to external tissues, showing a layer of collagen+ cell bodies internally followed by longitudinal muscle, then diagonal muscle, then circular muscle all strongly expressing TUBA-2, followed by nuclei in the epidermis. Animal was fixed with NAFA and detection of TUBA-2 and collagen mRNA was through sequential tyramide signal amplification of HRPconjugated antibodies (goat anti-rabbit-HRP for TUBA-2 and anti-digoxigenin-HRP for collagen). Scale bar, 10 microns.