Fixation permeabilization concentrate

Freshly collected dura tissue was sieved through a cell strainer, followed by centrifugation (1,500 rpm, 5 min) to prepare single-cell suspensions. Cells were incubated with antibodies against cannabinoid markers CB1, CB2 (Bioss Inc.), and TRPV1 (Novus Biologicals Ltd.). Following a PBS wash, cells were fixed and permeabilized using a Fixation/Permeabilization Concentrate (Biolegend, CA, USA), and then incubated with antibodies for intracellular labeling of MAGL, FAAH (Purchased from Bioss Inc.), and Cox-2 (Abcam). After a final wash, cells were analyzed using a 4-color flow cytometer (Acea 4-laser NovoCyte Quanteon, Agilent technologies lnc., USA), and analyzed using FCSexpress^™ software (De novo software, CA, USA), as described previously (Baban et al., 2005; Baban et al., 2013). Isotype-matched controls were analyzed to set the appropriate gates for each sample. For each marker, samples were analyzed in duplicate. To minimize false-positive events, the number of double-positive events detected with the isotype controls was subtracted from the number of double-positive cells stained with corresponding antibodies (not isotype control), respectively. Cells expressing a specific marker were reported as a percentage of the number of gated events.