Autoradiographic Mapping of Vasopressin Receptors

Whole body samples were cryosectioned in the sagittal plane in 8 series of 20 μm. Tissue was mounted on SuperFrost Plus slides (VWR) and stored at − 80 °C. Standard methods¹⁹ were used to perform receptor autoradiography with 0.05 nM ¹²⁵I labeled linear AVP receptor ligand (AVPR1A antagonist NEX310; NEN/Perkin-Elmer, Waltham, MA^{21 (link),22 (link)}). To competitively assess nonspecific binding in adjacent sets of slides from WT and AVPR1A KO mice, unlabeled AVP peptide (V9879, Sigma Aldrich) was added to half of the tracer buffer to yield two concentrations of unlabeled AVP competition (0 nM and 1000 nM). Autoradiographic films (Kodak Biomax MR film, Carestream Health, Inc., Rochester, NY, USA) were exposed to slides and ¹⁴C autoradiographic standards (ARC-0146; American Radiolabeled Chemicals, St. Louis, MO, USA) using previously established methods^{17 (link)} for one 3-day period and one 10-day period before developing (Mini-Medical/90 X-ray film processor, AFP Imaging, New York).