nimh.nih.gov/lcmr/snge/Protocols/ISHH/ISHH.html). Slides were exposed to Amersham Hyperfilm MP film for 2 months at room temperature with appropriate 14C-labeled standards (Amersham, Little Chalfont,

UK). No specific hybridization was detected with sense probes and no APJ mRNA signal above background was detected in tissues from APJ KO mice. Some slides were subsequently dipped in Ilford K5 nuclear emulsion and stored desiccated at 4 °C for 4–6 months before development using Kodak D19 at room temperature. Tissue sections were counterstained with toluidine blue. Mouse cryostat sections (20 μm) were cut and thaw mounted onto subbed (gelatin, vanadium oxide) slides. APJ receptor autoradiography was performed with Ku-0059436 modifications of the procedure described by Katugampola et al. [21]. Brain sections were fixed in 0.1% PFA in PBS for 5 min and rinsed in 10 mM Hepes pH 7.5.

All sections were pre-incubated for 20 min in 20 mM Hepes pH 7.5 containing 1 mM EDTA, 0.3% BSA and Sigma Protease Inhibitor Complex (Sigma, Dorset, UK). Slides were then incubated in 20 mM Hepes, pH 7.5, 100 mM NaCl, 5 mM Vincristine MgCl2, 10 mM KCl, 1 mM EDTA, Sigma protease inhibitor complex and 0.3% BSA containing radiolabeled (Glp65, Nle75, Tyr77) (125I)-Apelin-13 [0.5 nM] (Perkin Elmer, Cambridgeshire, UK) in the absence or presence of unlabeled (Pyr1)-apelin-13 [1 μM] (Bachem, Germany) as a displacer. Binding specificity was assessed by comparison of the distribution of [125I]-(Pyr1)apelin-13 binding sites in wildtype tissue to that in APJ KO tissue. Incubation lasted 1 h at RT in a humid chamber and was followed by 2 × 10 min washes in ice-cold 20 mM Hepes pH 7.5, 0.3% BSA with stirring

and 2 × 15 min washes in ice-cold 10 mM Hepes pH 7.5. Slides were then rinsed in ice-cold dH2O and air-dried fantofarone at 4 °C before being exposed to X-ray film (Amersham Hyperfilm MP) for 2 weeks. Following this some slides were re-exposed to emulsion-coated film (Amersham Hyperfilm 3H) for 1 month to obtain better macroscopic resolution. Films were developed as described for ISHH, except emulsion-coated films, which were developed manually as per manufacturer’s instructions. ISHH with antisense APJ riboprobes was used to map the distribution of APJ mRNA in the male and female mouse brain and peripheral tissues. Sections from all tissues were also hybridized with sense APJ riboprobes as controls and showed only background level of labeling. A number of tissues, including the pituitary, lung, heart, ovary and uterus, showed high levels of hybridization, with representative photographs shown in Fig. 1, Fig. 2 and Fig. 3. Within the brain APJ mRNA had a very restricted distribution where the PVN and SON hypothalamic regions showed high levels of gene expression (Fig. 1A and B). No labeling of other structures throughout the brain was observed.