Cdh11 antibody specificity and morpholino-induced expression reduction. A: Immunoblot analysis using anti-Cdh11 rabbit polyclonal antibody (see Experimental Procedures section) to probe adult zebrafish brain homogenate (Cdh11 was abundantly detected in the adult brain) reacted with a single polypeptide (relative migration ∼120 kD; right lane). Immunodetection was reduced by co-incubating the anti-Cdh11 antibody with the immunogen Cdh11 peptide (i.e., used to raise the antibody; middle lane), but immunodetection was not affected by co-incubating the anti-Cdh11 antibody with an identical concentration of an irrelevant peptide (right lane). Immunofluorescence using anti-Cdh11 rabbit polyclonal antibody in wholemount 30-hpf embryos. The typical inner ear Cdh11 expression pattern was detected in control embryos (B), but co-incubating the anti-Cdh11 antibody with the immunogen Cdh11 peptide eliminated all labeling (C). Laser power, gain settings, filters, and image processing were identical for B and C. D: Pools of protein homogenate from three embryos (24 hpf) per lane were immunoblotted to detect Cdh11. This was the smallest number of embryos in which we could reliably detect Cdh11 expression in control embryos (left lane). A pool of three cdh11 morpholino oligonucleotide--injected 24-hpf embryos showed reduced Cdh11 expression levels. A representative result from two experiments is shown.
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