To begin to address the significance of PAR2 to myelination of the spinal cord, we quantified the expression of PAR2 RNA in the spinal cord of mice at P0, 7, 21 or 45 using real time PCR (Radulovic et al., 2015 (link)). RNA was isolated using RNA STAT-60 (Tel-Test, Friendswood, TX) and stored at -70 C until the time of analysis. Amplification of the housekeeping gene 18S in the same RNA samples was used to control for loading. Real-time PCR amplification in each case was accomplished using primers obtained from Integrated DNA Technologies (Coralville, IA), or Applied Biosystems (Grand Island, NY), as detailed in Table 1, on an iCycler iQ5 system (BioRad, Hercules, CA). In addition, we localized PAR2 (sc-8205, RRID: AB_2101309, Santa Cruz, Santa Cruz, CA) to Olig2 (Ab9610, RRID:AB_10141047, Millipore, Temecula, CA) or CC-1-postive cells (adenomatous polyposis coli, OP80, RRID:AB_213434, Millipore, Temecula, CA) within the spinal cord white matter at the P21 peak of myelination, using immunofluorescence techniques. Stained sections were cover slipped with Hardset containing DAPI (Vector, Burlingame, CA) and digitally imaged (Olympus BX51 microscope, Olympus, Center Valley, PA). Counts were made of either Olig2 or CC-1+ cells with a DAPI stained nucleus within the entire dorsal column of at least 3 mice at each time point without knowledge of genotype.