All chemicals used were purchased from Sigma (St. Louis, MO)
unless otherwise indicated. Formaldehyde was obtained from
Polysciences, Inc. (Warrington, PA), the microscope slides were
Colorfrost/Plus from Fisher Scientific (Pittsburgh, PA), and the
optimal cutting compound (OCT) from Sakura (Tokyo, Japan).
Helix
pomatia (HPA) and
Ulex europaeus-I (UEA-I) agglutinins
conjugated to Texas red and tetramethylrhodamine isothiocyanate
(TRITC), respectively, were obtained from Sigma. Rabbit polyclonal
antibodies to the following nerve markers and receptors were purchased:
protein gene product (PGP) 9.5 (Accurate Chemical and Scientific
Corporation, Westbury, NY), vasoactive intestinal peptide (VIP; Dia
Sorin, Stillwater, MN), dopamine β-hydroxylase (DBH; Eugene Tech
International, Inc., Ridgefield Park, NJ), tyrosine hydroxylase (TH;
Calbiochem-Novabiochem Corp., San Diego, CA),
M
1-, M
2-, and
M
3-muscarinic receptor subtypes (R & D
Antibodies, Berkeley, CA), and α
1A-,α
1B-, α
1C-,β
1-, β
2-, andβ
3-adrenergic receptor subtypes (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA). Two different antibodies (raised
either in mouse or human) against α
1A orβ
3-adrenergic receptors were used for both
immunohistochemistry and Western blotting. The source, receptor subtype
specificity, and species specificity for the primary antibodies to
adrenergic receptors are summarized in
Table 1 . The secondary antibodies for immunofluorescence experiments
were fluorescein isothiocyanate (FITC)-conjugated and were purchased
from Jackson Laboratories (West Grove, PA). The secondary antibodies
for Western blotting were horseradish peroxidase (HRP)-conjugated
anti-IgG and purchased from Santa Cruz Biotechnology, Inc. Vectashield
mounting media, with or without DAPI, were from Vector Laboratories
(Burlingame, CA). All the reagents for Western blotting were purchased
from Bio-Rad Laboratories (Hercules, CA), and the chemiluminescence
reagents for visualization from Pierce (Rockford, IL).