Mouse anti kir6

For immunofluorescent staining of the aortic tissue sections, we used sodium citrate (0.01 M, pH 6.0) for antigen retrieval. Sections were then blocked for 30 min with 5% BSA in PBS at room temperature. The following diluted primary antibodies: mouse anti-BK_Ca α (1:200, Abcam, Cambridge, MA, UK), rabbit anti-BK_Ca β (1:1000, Abcam, Cambridge, MA, UK), rabbit anti-Kir6.1 (1:200, Santa Cruz, CA, USA), mouse anti-Kir6.2 (1:50, Santa Cruz, CA, USA), rabbit anti-Ca_v1.2 (1:100, Santa Cruz, CA, USA), goat anti-Ca_v1.3 (1:200, Santa Cruz, CA, USA), were used to incubate sections overnight at 4 °C. Slides were then incubated at 37 °C with secondary antibodies Alexa Fluor 488 (donkey anti-goat, 1:400; Life Technologies), Alexa Fluor 488 (donkey anti-rabbit, 1:400; Life Technologies), Alexa Fluor 488 (donkey anti-mouse, 1:400; Life Technologies), Alexa Fluor 594 (donkey anti-rabbit, 1:400; Life Technologies), Alexa Fluor 594 (donkey anti-mouse, 1:400; Life Technologies) and Alexa Fluor 594 (donkey anti-goat, 1:400; Life Technologies) for 1 h. 4′,6-diamidino-2-phenylindole (DAPI) (blue) was used to counterstain all sections. The slides were then mounted and viewed under a fluorescent microscope (Olympus BX53).

The thoracic aorta tissue sections were incubated with 0.3% H₂O₂ to quench endogenous peroxides. The sections were boiled in sodium citrate (0.01 M, pH 6.0) for antigen retrieval. After 10-minute-permeation with 0.2% Triton X-100, they were blocked for 30 min with 5% bovine serum albumin (BSA) in PBS at room temperature. The following diluted primary antibodies: mouse anti-BK_Ca α (1:200, Abcam, Cambridge, MA, UK), rabbit anti-BK_Ca β (1:1000, Abcam, Cambridge, MA, UK), rabbit anti-Kir6.1 (1:200, Santa Cruz, CA, USA), mouse anti-Kir6.2 (1:50, Santa Cruz, CA, USA), rabbit anti-Ca_v1.2 (1:100, Santa Cruz, CA, USA), goat anti-Ca_v1.3 (1:50, Santa Cruz, CA, USA), rabbit anti-endothelial nitric oxide synthase (eNOS) (1:500, Wuhan GoodBio Technology CO., Ltd. Wuhan, China), were used to incubate sections overnight at 4 °C. The slides were then incubated at 37 °C with biotinylated immunoglobulins and avidin-biotin peroxidase complex after washing with PBS, while H₂O₂ (3%) and diaminobenzidine tetrahydro chloride (DAB, 5 mg/10 mL) (Sigma-Aldrich, St. Louis, MO, USA) were used as chromogens to visualize reaction products. The immunostaining results were examined under a DM 4000B Microscope (Leica), and the average optical intensities of the relevant proteins were determined using Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA).