Five new anti-zebrafish cytochrome P450 (CYP) antipeptide polyclonal antibodies were tested for tissue specificity using immunohistochemistry. The antibodies are for the zebrafish CYPs IA, 2K6, 2K7, 3A65 and 3C1. Three of these antibodies were also compared to existing antibodies made against CYP isozymes in rainbow trout (Oncorhynchus mykiss). Individual staining protocols for juvenile zebrafish tissue were optimized. Of the antibodies tested, two required a non-crosslinking fixative, such as 70% ethanol, for best specificity. All except one achieved better results when incubated overnight at a higher dilution. Overnight incubation decreased background staining as well as conserving antibodies. Several antigen retrieval methods were explored, but none with better results than extended incubation. Localization of expression of each CYP was recorded qualitatively. Results showed a near lack of expression of all five zebrafish CYPs tested in the liver, the organ where most xenobiotic metabolism occurs. Other noteworthy results include multiple layers of eye tissue expressing CYP3C 1 and intense stain in the muscle layer of the entire intestine with the anti-CYP2K6 antibody. An antibody for CYP3A27, the trout ortholog of zebrafish CYP3A65, stained the anterior portion of the intestine, the location of intestinal tumors. Nearly every antibody showed some enzyme presence in the intestine, skin and pharynx.
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