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.