Simultaneous detection of neuropeptides and messenger RNA in the magnocellular hypothalamo-neurohypophysial system by a combination of non-radioactive in situ hybridization histochemistry and immunohistochemistry.
A protocol was developed combining non-radioactive in situ hybridization histochemistry with enzyme based immunohistochemistry, detect the expression of mRNA in phenotypically defined neurons. Free-floating brain sections were hybridized with the oligonucleotide probes which have been 3'-end labelled with biotin-11-dUTP. The hybridized probe was visualized by a combined avidin-biotin bridge method, anti-avidin immunohistochemistry, and horseradish peroxidase detection using diaminobenzidine as a substrate. The in situ hybridization step yielded a very stable reaction product enabling subsequent immunohistochemical reactions using horseradish peroxidase and benzidine dihydrochloride as a chromogen. Magnocellular neurons of the hypothalamo-neurophypophysial system synthesize either vasopressin or oxytocin; water deprivation and chronic saline ingestion are potent stimuli for the expression of both of the genes encoding these neuropeptides. A number of other neuropeptides with putative transmitter action are synthesized in magnocellular neurons during such stimulation. Experiments were performed to explore whether neuropeptide Y immunoreactivity is present within magnocellular vasopressin mRNA-expressing neurons of the hypothalamo-neurophypophysial system. The results clearly demonstrated that neuropeptide Y-immunoreactive elements were present within a number of magnocellular vasopressin mRNA-containing cells. In addition, immunohistochemical detection of the neuropeptides ocytocin and cholecystokinin was carried out on sections hybridized non-radioactively for vasopressin; as expected vasopressin mRNA did not co-exist with cholecystokinin, whereas a few oxytocin immunoreactive neurons in osmotically stimulated animals also contained vasopressin mRNA. The developed method makes possible the immunohistochemical detection of intracellular antigens with concomitant detection of intracellular mRNA.
Larsen
P J
PJ
Department of Anatomy, University of Copenhagen, Denmark.
Mikkelsen
J D
JD
D000818:Animals; D000941:Antigens / Q000032:analysis; D001127:Arginine Vasopressin / Q000235:genetics; D015342:DNA Probes; D007030:Hypothalamo-Hypophyseal System / Q000737:chemistry* / Q000166:cytology; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008297:Male; D009411:Nerve Endings / Q000737:chemistry; D009474:Neurons / Q000737:chemistry; D009478:Neuropeptide Y / Q000032:analysis; D009479:Neuropeptides / Q000032:analysis*; D009841:Oligonucleotides; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity
D000941:Antigens; D015342:DNA Probes; D009478:Neuropeptide Y; D009479:Neuropeptides; D009841:Oligonucleotides; D012333:RNA, Messenger; D001127:Arginine Vasopressin