Alexa fluor secondary antibody

Paraffin-embedded atrial tissue sections from five patients per group were de-paraffinized in xylene, then rehydrated in graded ethanol and phosphate-buffered saline solution (PBS) and the antigen was unmasked using sodium citrate (10 mmol/L, pH = 6.0) at 90-100°C for 30 minutes. The samples were then washed using more PBS and blocked with 2% bovine serum albumin in PBS for 2 hours at room temperature. After another PBS wash, sections were incubated overnight with anti-alpha-1A and anti-alpha-1B antibodies (each used at 1.1μg/ml); Abcam) and smooth muscle actin antibody (2.5μ/ml) at 4°C as in previous studies.^{11 (link), 22 (link), 23 (link)} Sections were then washed in PBS and incubated with the applicable Alexa-fluor secondary antibodies and mounted using a fluorescent mounting medium (Vector Labs, Burlingame, CA). Fluorescent microscopy was conducted using a Zeiss LSM510 confocal microscope system (Carl Zeiss MicroImaging, Inc., Thornwood, NY) in a similar fashion to previous studies.^{23 (link)-25 (link)} Negative controls were produced by labeling tissues with only the anti-actin antibodies along with the secondary antibodies.

Atrial tissue sections from five patients per group were first de-paraffinized in xylene then rehydrated using graded ethanol and phosphate-buffered saline solutions (PBS). The antigen was unmasked using sodium citrate (10mmol/L, pH = 6.0), followed by a PBS wash and blocking with 2% bovine serum albumin in PBS at room temperature for 2 hrs.^{4 (link)–6 (link)} After PBS wash, sections were incubated overnight with anti-V1A and V1B antibodies (each used at 1:200) (ABCAM, Cambridge, MA) and smooth muscle actin antibody at a temperature of 4°C. Sect ions were then washed in PBS and incubated with the appropriate Alexa-fluor secondary antibodies and mounted using fluorescent mounting medium (Vector Labs, Burlingame, Calif.). Tissue was visualized using a Zeiss LSM510 confocal microscope system (Carl Zeiss MicroImaging, Inc. Thornwood, NY). Tissue labeled with only the actin antibody and secondary antibody served as a negative control.

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)