2.8 Immunohistochemistry
To assess the distribution of MOPs in the DRG and spinal cord, we
performed immunofluorescence imaging. Animals, aged 6–8 weeks, were
deeply anesthetized through isoflurane inhalation under saturated vapor
pressure at room temperature, and perfused intracardially with 0.1 M
phosphate-buffered saline (PBS; pH 7.4, 4°C) followed by fixative (4%
formaldehyde and 0.2% [v/v] picric acid in PBS, 4°C). Tissues were
cryoprotected in 30% sucrose for at least 24 hr before being serially
cut into 15-μm sections and placed onto slides. Sections of spinal cord
and lumbar DRG were cut on a cryostat and immunostained with guinea pig
anti-MOP antibody (Neuromics, Cat
#GP10106,
RRID:AB_2737108, 1:400). For secondary antibodies, we used Alexa
488-conjugated goat antibody to mouse (Thermo Fisher Scientific, Cat
#A-10667,
RRID: AB_2534057) and Cy3-conjugated goat antibody to guinea pig
(Thermo Fisher Scientific, Cat
#A-11073,
RRID:AB_2534117). All secondary antibodies were diluted 1:100 in
blocking solution (PBS + 1% bovine serum albumin [BSA] + 0.1%
Triton X-100). Images were taken at 40x with an AXIO Examiner.Z1
confocal microscope (Carl Zeiss AG, Oberkochen, Germany). Tissues from
both experimental groups were processed simultaneously during the
immunohistochemical process.