Biology Reference
In-Depth Information
3.2 Materials and Methods
The RG was dissected along its entire length in three suckling lambs, 1-3 days of
age, and separated into proximal (the half next to the cardia) and distal (the half next
to the reticulum-omasum orificium) parts. The specimens were fixed in 4% para-
formaldehyde in phosphate buffer (0.1 M, pH 7.2) at 4
C for 24 h, then rinsed in
phosphate-buffered saline (PBS), and stored at 4
C in PBS containing 30% sucrose
and sodium azide (0.1%). The samples were frozen in isopentane cooled in liquid
nitrogen. Transverse (i.e., perpendicular to the floor of the groove) cryosections
(20
m) were obtained along the entire length, mounted on poly-
L
-lysine-coated
slides and submitted to double indirect immunohistochemical methods using the
antibodies listed in Table
3.1
. The sections were examined by a Zeiss Axioplan
microscope equipped with appropriate filter cubes. The percentages of the NOS-
immunoreactive (IR) neurons were calculated in nonconsecutive sections using
NOS/NSE stain, and at least 100 cells were counted both in the lips and in the floor
of each subject. For double-stained NOS/GAL and NOS/DBH, at least 50 neurons
were counted for each subject both in the lips and in the floor. The values of the
lip-floor junctions were included in the lip count.
m
3.3 Results
NSE-IR nerve fibers were observed along the entire length of the RG, and there was
no obvious gradient of innervation between the proximal and distal parts. The
neurons were generally clustered in ganglia varying in size; large ganglia (up to
Table 3.1 List of antibodies used in this study
Neuronal markers
Species
Code
Dilution
Supplier
NSE
Rabbit
N1516
1:5
DAKO
NOS
Mouse
Sc-5302
1:200
Santa Cruz
NOS
Rabbit
SA-227
1:300
Biomol
GAL
Rabbit
IHC 7153
1:400
Peninsula
DBH
Mouse
MAB308
1:200
Chemicon/Millipore
TH
Mouse
MONX10786
1:60
Monosan
SP
Rat
10-S15A
1:400
Fitzgerald
CGRP
Rabbit
C8198
1:800
Sigma-Aldrich
CALB
Rabbit
CB-38
1:800
Swant
NF200 kDa
Rabbit
N 4142
1:1000
Sigma-Aldrich
IB4
—
L2140
1
g/mL
Sigma-Aldrich
m
Secondary antisera
FITC-conjugated donkey anti-mouse IgG
1:100
Jackson
TRITC-conjugated donkey anti-rabbit IgG
1:500
Jackson
Alexa 594-conjugated donkey anti-rat IgG
1:400
Mol. Probes
FITC-conjugated streptavidin
1:100
Vector
