E800 epi fluorescent microscope system

Skeletal muscle sections were de-paraffinized in xylene, rehydrated in graded ethanol and phosphate-buffered saline solution, and antigen-unmasked with sodium citrate (10 mmol/L, pH = 6.0) followed by phosphate-buffered saline (PBS) wash and blocking with 2%bovine serum albumin in phosphate-buffered saline at room temperature for 2 hours.^{17 (link),18 (link)} After phosphate-buffered saline wash, overnight incubation with anti- CD3 (Cell Signaling), VE-cadherin (Cell Signaling), and anti-phospho-VE-cadherin (Lifespan Biosciences Inc. Seattle, WA) antibodies (each used 1:50) was performed at 4°C. Anti-mouse, smooth muscle actin (1:1000; Sigma, St Louis, Mo) was used to detect microvascular smooth muscle. Sections were then washed in phosphate-buffered saline and incubated with the appropriate Alexa fluor secondary antibody and mounted using fluorescent mounting medium (Vector Labs, Burlingame, Calif). Tissue labeling with secondary antibody alone or with normal rabbit IgG or serum in place of primary antibody served as a negative control. Tissue was visualized using a Nikon E800 epi-fluorescent microscope system (Nikon Inc. Melville, NY). Six image photos per slide were taken at same magnificent resolution (20 x Plan Fluor objective) for optical density (intensity) analysis (Spot RT3, Diagnostic Instruments, Sterling Heights MI)

Atrial tissue sections were deparaffinized in xylene, rehydrated in graded ethanol and PBS, and antigen-unmasked with sodium citrate (10 mmol/L, pH = 6.0) followed by PBS wash and blocking with 2% bovine serum albumin in PBS at room temperature for 2 hours [6 –9 , 11 (link), 12 (link)]. After the PBS wash, overnight incubation with anti-COX-2, NOX-2, and NOX-4 (ABCAM) was performed at 4°C. Antimouse, smooth muscle actin (Sigma-Aldrich, St Louis, MO) was used to detect microvascular smooth muscle. Sections were then washed in PBS and incubated with the appropriate Alexa Fluor secondary antibody (Thermo Fisher Scientific) and mounted using Fluorescent mounting medium (Vector Laboratories). Tissue was visualized using a Nikon E800 epiFluorescent microscope system (Nikon). Six image photos per slide were taken at the same magnification resolution (×20 Plan Fluor [Nikon] objective) for optical density analysis (Spot RT3; Diagnostic Instruments).