2.2 Recording of vasomotor responses
The biopsies were stored for 16 h at 4 °C in 30 mL PSS to wash away anesthetics and analgesics(Batenburg, Garrelds, van Kats, Saxena & Danser, 2004). They were then transferred to PSS at room temperature (RT, 22⁰C) and one long arterial resistance segment (6-12 mm) was isolated from the pericardial sheet, cleaned from connective tissue, and divided into 2 mm segments. These preparations were mounted in multi-chamber wire myographs (DMT 620M, Danish Myo Technology, Aarhus, Denmark) to record isometric tension development. The organ chambers contained 5 mL PSS maintained at 37 °C and continuously aerated with 5% CO2 in air. Prior to pharmacological analysis, segments were stretched to a lumen diameter and resting wall tension corresponding to a transmural pressure of 100 mmHg according to the law of Laplace. We previously observed that under these conditions this type of arterial preparation developed the strongest contractile responses (Leurgans et al., 2016). After 5-7 hours of pharmacological study, the distended arterial segments were fixed by overnight incubation in 4% formaldehyde solution at RT and then stored in PBS/0.05% sodium-azide at 4⁰C for subsequent immunohistochemical analyses.