Detection of mRNA encoding Crustacean Hyperglycemic Hormone (CHH) in the eyestalk of the crayfish Orconectes limosus using non-radioactive in situ hybridization

A non-radioactive in situ hybridization procedure for the localization of the mRNA encoding the crustacean hyperglycemic hormone (CHH) in the eyestalk of the crayfish Orconectes limosus has been developed. Based on the partial amino acid sequence of CHH, polymerase chain reactions were performed to generate complementary DNA (cDNA) clones encoding CHH. Non-radioactively labelled probes derived from the cDNA sequence were used to establish suitable conditions in terms of tissue fixation and pretreatment for detection of the CHH-encoding mRNA in combination with an immunocytochemical staining using a polyclonal antibody for CHH. Localization of the mRNA in the CHH perikarya was obtained with a complementary RNA probe in combination with pepsin/HCl treated Bouin-fixed eyestalks. The immunocytochemical staining confirmed that this cRNA probe specifically hybridized with mRNA of cell somata belonging to the CHH-producing cell system in the eyestalk of Orconectes limosus.