Immunohistochemistry-Frozen: Substance P Antibody (NC1/34) [NB100-65219] - Opioid R/OPRM1 (green) and Substance P (red) were detected in frozen mouse spinal cord and DRG tissue by fluorescent multiplex IHC. Mu Opioid R/OPRM1 Antibody (NB100-1620), used at 1:100, and Substance P Antibody (NB100-65219), used at 1:50, were applied to the tissue and simultaneously incubated at 4C overnight. Anti-Rabbit Alexa Fluor(R) 488-conjugated secondary antibody and anti-Rat NL557-conjugated secondary antibody (NL013) were applied to the tissue, and simultaneously incubated at room temperature, protected from light for 1 hour. Coverslips were applied to the tissue slide along with mounting media containing DAPI (blue) and imaged using a 4X objective lens.