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Fig. 1 ZCRDH is expressed in zebrafish cone inner segments An antibody was generated against an N-terminal peptide ZCRDH (product of the zgc:153441 gene) ( Figure S2 ). (A) The antibody was tested with four different protein homogenates: pcDNA3.1 HEK, HEK293T cells transfected with the non-recombinant expression plasmid, pcDNA3.1; ZCRDH HEK, HEK293T cells transfected with plasmid containing the ZCRDH coding region; wild-type ZF eyes, whole-eye tissues from wild-type (AB strain) zebrafish; and Zcrdh-mutant eyes, whole-eye tissues from Zcrdh-mutant zebrafish. The antibody detected a protein band of the predicted size (∼37 kDa) in retinal homogenates from wild-type and ZCRDH-expressing HEK293T cells. The slightly higher molecular mass of ZCRDH in HEK-cell versus wild-type zebrafish retina homogenates is due to the presence of a FLAG epitope tag at the C terminus of recombinant ZCRDH. The same blot was re-probed with an antibody against β-tubulin as a protein loading control, shown below the ZCRDH immunoblot. A similar blot was probed with the ZCRDH antibody after preincubation with the immunizing peptide. Note the absence of signal in this control for Ab specificity. (B) Immunohistochemistry performed on adult wild-type zebrafish retinal sections using ZCRDH Ab (red), peanut agglutinin (PNA) lectin to label the sheath surrounding cone outer segments (green), and the nuclear stain 4′,6-diamidino-2-phenylindole DAPI (blue). Scale bar, 20 μm. ZCRDH immunoreactivity is strongly present in cone inner segments. (C) Immunocytochemistry performed on adult Zcrdh-mutant retinal sections using the same ZCRDH Ab, PNA, and DAPI labels as in (B). The region labeled “rod and cone IS and OS” corresponds to the region of rod and cone photoreceptor inner and outer segments, respectively. The region labeled “ONL” corresponds to the outer nuclear layer containing rod and cone photoreceptor nuclei. Scale bar, 20 μm. Note the greatly reduced labeling of cone inner segments in Zcrdh-mutant versus wild-type zebrafish retinas. See also Figure S2 .