Fluorescent whole-mount immunohistochemical detection of myocilin in the eye of zebrafish embryos (96hpf). Wild-type (A–C), heterozygous (D–F) and homozygous (G–I) myoc mutant embryos were incubated with a chicken conjugated goat anti-chicken IgY secondary antibody. Three-dimensional reconstruction from z-stack scanned confocal microscopy images (A,D,G) of the eye. Optical sections (92 μm) 8 (B,E,H) and 16 (C,F,I), from the exterior ocular surface, were selected from z-stack images to show the precise localisation of the green signal in the external and internal surface of the optic cup (white arrows and yellow arrowheads, respectively), lens epithelium (white arrowhead), and dorsoposterior and ventral periocular tissues (yellow arrowheads) (A–C). Blue: DAPI nuclear staining. Green: Cy2-conjugated goat anti-chicken IgY secondary antibody. Red: tissue autofluorescence. The cross indicates the position of the embryonic axes (D: dorsal; P: posterior; V: ventral; A: anterior). The images are representative of the result observed in 10 embryos. le: lens. re: retina. The negative controls are shown in Figure S3A–C. Two-dimensional confocal image z-stacks are shown in Supplementary Video S1.
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