In each case, at least one series of brain sections was
processed for detection of PRV-Ba. Additional series were processed for
detection of both PRV-Ba and TH or of PRV-Ba and ChAT in the same
sections. TH was used as a marker of the A5 noradrenergic cell group.
ChAT is a marker of cholinergic neurons. Sections were circled with a
hydrophobic slide marking pen (PAP pen; Electron Microscopy Sciences,
Fort Washington, PA) and briefly dried on a slide warmer. The steps for
detection of PRV-Ba were as follows: (1) Rinse slides in 0.02 M
phosphate-buffered saline (PBS) with 0.1% sodium azide
(NaN3; Sigma, St. Louis, MO) two times over 30
minutes on a rotary shaker; (2) quench endogenous peroxidases with 10%
methanol and 3% hydrogen peroxide in PBS for 5 minutes on a rotary
shaker; (3) rinse in PBS three times over 30 minutes on a rotary
shaker; (4) block with 2% nonfat dry milk and 0.3% Triton X-100
(Sigma) for 1 hour; (5) incubate in primary antibody (goat anti-PRV-Ba
generated by two of the authors [PR, MSL]; 1:100,000), 3% rabbit
serum, and 0.1% Triton in PBS-NaN3 overnight;
(6) rinse in PBS three times over 30 minutes on a rotary shaker; (7)
incubate in secondary antibody (biotinylated rabbit anti-goat (1:500;
Vector Laboratories, Burlingame, CA), 2% rabbit serum, and 0.1%
Triton in PBS-NaN3 for 4 hours; (8) rinse in PBS
three times over 30 minutes on a rotary shaker; (9) incubate in reagent
(Elite ABC; Vector Laboratories) and 0.1% Triton in PBS for 90 minutes
at room temperature; (10) rinse in PBS three times over 30 minutes on a
rotary shaker; (11) subject to a nickel-intensified diaminobenzidine
(black) reaction; and (12) rinse, air dry, dehydrate, clear, and
coverslip. In double-staining for PRV-Ba (nickel-intensified
diaminobenzidine, black) and tyrosine hydroxylase (diaminobenzidine
without nickel intensification, brown) on the same sections, slides
were rinsed for 30 minutes after step 11 and then processed starting
with step 5, using monoclonal antibody 318 (1:1000; Chemicon
International, Temecula, CA).
Because initial experiments that used double-label immunohistochemistry
for PRV-Ba and TH with diaminobenzidine substrates demonstrated very
close spatial relationships between PRV-Ba- and TH-immunoreactive (IR)
neurons
(Fig. 1G) , fluorescent-tagged secondary antibodies and confocal
microscopy were used in subsequent experiments to evaluate the
possibility that some parasympathetic preganglionic neurons within the
SSN express TH. After incubation with primary antibodies, a
cyanine-tagged donkey anti-goat antibody (Cy2; Jackson ImmunoResearch
Laboratories, West Grove, PA) and an indocarbocyanine-tagged donkey
anti-mouse antibody (Cy5; Jackson ImmunoResearch Laboratories) were
used at 1:250 dilutions to detect PRV-Ba and TH, respectively.
For PRV-Ba and ChAT double-labeling, a rabbit polyclonal antibody to
ChAT (gift from Miles Epstein, University of Wisconsin, Madison) was
used at a 1:500 dilution and the goat anti-PRV-Ba antibody was used at
a 1:50,000 dilution. Secondary antibodies, Cy2-tagged donkey anti-goat
and Cy5-tagged donkey anti-rabbit, were used at 1:250 dilutions. Tissue
was incubated with secondary antibody for 3 hours, rinsed, dehydrated,
cleared, and coverslipped with 1,3-diethyl-8-phenylxanthine mounting
compound (DPX; Sigma). Fluorescence was visualized with a confocal
laser-scanning microscope (Bio-Rad, Hercules, CA).
Orbital tissues, the trigeminal nerve, GPN, and PPG were processed for
detection of PRV-Ba, CTB, or fast blue. For detection of CTB, a goat
polyclonal antibody was used (1:30,000; List Biological Laboratories)
in the sequence of steps described for PRV-Ba. Fast blue was visualized
with an epifluorescence microscope equipped with ultraviolet filters
(Axioplan; Zeiss, Oberkochen, Germany). In two rats (four eyes), fast
blue was the only tracer used. Every section was collected, and labeled
cells in the PPG and along the GPN were counted. Care was taken to
avoid counting individual fast blue–labeled ganglion cells that
appeared on adjacent sections more than once.