Axio observer a1 obj 20x

Production of NO was assessed as previously described [19 (link)]. HB (100 µg/mL) or acetylcholine (10−6 M) was administered to the mesenteric artery in the last 30 min of 4-amino-5-methylamino-2,7,-diflfluoroflfluorescein diacetate (DAF-FM) incubation, alone and after 20 min exposure to L-NAME (300 μmol/L, 30 min). Mesenteric segments were cut in 5-µm thick sections, observed under a fluorescence microscope (Zeiss Axio Observer A1—Obj. 20X), subsequently counterstained with haematoxylin and eosin, and observed under a light microscope.

Dihydroethidium (DHE, Life Technologies) was used to evaluate production of reactive oxygen species (ROS) in mouse mesenteric arteries, as previously described [20 (link)]. Briefly, vessels were incubated with 5 µM of DHE for 20 min and subsequently observed under a fluorescence microscope (Zeiss Axio Observer A1—Obj. 20X). Images were acquired by a digital camera system (Olympus Soft Imaging Solutions). A second, estimation of total ROS production in mouse vessels was performed with the membrane-permeable fluorescent probe an analog of 2,7-dichlorodihydrofluorescein (DCDHF), dihydrorhodamine 123 (DHR123) (Invitrogen). After treatment, vessels were incubated with Krebs solution containing 5 μM DHR123 for 30 min at 37 °C, and then washed two times with PBS prior to fluorescence measurement using a fluorescence microplate reader (TECAN infinite 200 Pro).