Sympathetic neurons or neurons from the nodose ganglia of the vagal nerve were plated on collagen-coated glass coverslips in 24 well plates. To stain living cells, human IgG was added (0.1 mg/mL) for 3 hours before cells were fixed in paraformaldehyde, 4%, for 30 minutes and blocked with bovine serum albumin, 5%, in phosphate-buffered saline for 1 hour. Subsequently, cells were developed with fluorescein isothiocyanate–conjugated specific anti-human IgG (1:500) for 2 hours. For double staining, the fixed cells were blocked and incubated overnight at 4°C with antibodies against tyrosine hydroxylase (1:1000; rabbit; Chemicon, Temecula, California), vesicular acetylcholine transporter (1:1000; goat, Chemicon), or β-III-tubulin (1:1000; mouse; Promega, Madison, Wisconsin) before they were developed with fluorescence-labeled specific secondary antibodies. Coverslips were mounted and images were acquired using a fluorescence microscope (Carl Zeiss, Jena, Germany). Cocultures of sympathetic neurons and cardiomyocytes were fixed in paraformaldehyde, 4%, for 30 minutes, blocked with bovine serum albumin, 5%, in Triton X, 0.1%, and incubated overnight with antibodies against tyrosine hydroxylase (1:1000), β-III-tubulin (1:1000), synaptophysin (1:500; rabbit, Dako, Glosturp, Denmark), or α-actinin (mouse, 1:1000). After washing, the cultures were developed with fluorescence-labeled specific secondary antibodies, and images were acquired using an inverted fluorescence microscope.