Movie 3.

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 external to internal tissues, showing some diagonal muscle, then longitudinal muscle, and image is oriented diagonally with the anterior located at the bottom right. Animal was fixed with NAFA and detection of TUBA-2 and collagen mRNA was through sequential tyramide signal amplification of HRP-conjugated antibodies (goat anti-rabbit-HRP for TUBA-2 and anti-digoxigenin-HRP for collagen). Scale bar, 10 microns.