A rapid and sensitive radioimmunohistochemical assay for quantitation of vasopressin in discrete brain regions with an anatomical resolution.
Radioimmunoassay has become a widely used method to study different neuroactive substances from brain tissue extracts, but cannot provide anatomical resolution. Here we describe a simple and sensitive radioimmunohistochemical assay (RIHA) to quantify a peptide, vasopressin (VP), in discrete brain regions of rats with 3-day water deprivation. After decapitation, brains were removed, frozen with dry ice and cut into 14-microns cryostat sections which were then fixed with 4% paraformaldehyde in phosphate-buffered saline. After rinses, tissue sections were stored in a freezer until use. For RIHA, brain tissue sections were pre-incubated, and then incubated with rabbit vasopressin antibody (1:2000 dilution) for 24 h at room temperature. After rinses, sections were incubated with 125I-labeled goat antirabbit IgG (1:200 dilution) for 1 h. Specimens were processed for quantitative autoradiography after rinses and drying. RIHA with aid of a computer-assisted image analysis system revealed that the VP content was significantly reduced in the paraventricular hypothalamic nucleus (PVN) and supraoptic nucleus (SON) of rats with 3-day water deprivation, whereas a parallel in situ hybridization study further demonstrated that VP mRNAs in the PVN and SON were greatly increased. In summary, this experiment demonstrates that RIHA is a simple and powerful tool which is able to detect changes of VP in the hypothalamus of dehydrated rats. Combining this method with in situ hybridization to assess mRNA expression allows assessment of the functional significance of the peptide changes. In this case, dehydration depletes vasopressin and upregulates its synthesis. Therefore, the combined use of RIHA and in situ hybridization should have general applicability to evaluate the functional role of a peptide or neurotransmitter system in response to stimuli in a quantitative way with anatomical resolution.
Hwang
B H
BH
Department of Anatomy, Indiana University School of Medicine, Indianapolis 46202.
Wang
G M
GM
D000818:Animals; D001345:Autoradiography; D001921:Brain / Q000378:metabolism*; D005557:Formaldehyde; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry / Q000379:methods*; D017403:In Situ Hybridization; D007457:Iodine Radioisotopes; D008297:Male; D011108:Polymers; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity; D013997:Time Factors; D014018:Tissue Distribution; D016707:Tissue Fixation; D014667:Vasopressins / Q000235:genetics / Q000378:metabolism*; D014868:Water Deprivation / Q000502:physiology
D007457:Iodine Radioisotopes; D011108:Polymers; D012333:RNA, Messenger; D014667:Vasopressins; D005557:Formaldehyde; C003043:paraform