This article describes the uses of immunostaining in the diagnosis of cholestasis. To immunostain for bile salt export pump (BSEP) and multidrug resistance protein 3 in severe hepatobiliary disease manifest early in life can rapidly identify whether sequencing of ABCB11 or ABCB4 is likely to yield a genetic diagnosis. To immunostain for canalicular ectoenzymes as well as transporters, with transmission electron microscopy, can suggest whether sequencing of ATP8B1 is likely to yield a genetic diagnosis. Demonstrating BSEP expression can direct attention to bile acid synthesis disorders. Immunostaining for multidrug resistance-associated protein 2 serves principally as a control for adequacy of processing.