Enzymatic cleavage prior to antibody incubation as a method for neuropeptide immunocytochemistry

Abstract

When deplasticized Epon sections were treated with endo- and/or exopeptidases prior to incubation with antibodies, the neuropeptide immunoreactivity of secretory nerves was often altered in a predictable way. Cleavage of neurosecretory material in octopus nerves by trypsin and carboxypeptidase-B enhanced enkephalin-like immunoreactivity, while Molluscan neuropeptide-like immunoreactivity was prevented by tryptic cleavage. The enzyme effects indicated the occurrence of a heptapeptide (Tyr-Gly-Gly-Phe-Met/Leu-Arg-Phe) that contains both the enkephalin and the Molluscan neuropeptide sequence. Vasopressin terminals of the rat neurohypophysis, which presumably contain enkephalin precursor sequences, exhibited enkephalin-like immunostaining after tryptic cleavage. ACTH/β-endorphin cells of the rat intermediate pituitary, which synthesize the enkephalin sequence at the N-terminus of β-endorphin, exhibited enkephalin-like immunoreactivity when sections were treated with α-chymotrypsin or trypsin, but not after incubation with leucine-aminopeptidase or carboxypeptidase-B. Enkephalin-like immunostaining could not be induced in any way in ACTH/β-endorphin cells of the anterior pituitary. Enzymatic cleavage may give additional information in immunocytochemical localization studies on neuropeptide sequences in secretory nerves and hormonal granules.