Fig. 4

Immunohistochemical analysis of the AEA system. A Immunohistochemistry of naïve male and female V1M cortex. Staining for NAPE-PLD, FAAH, Iba1, GFAP, and Nissl was performed. Original magnification ×63. B Immunohistochemistry of naïve male and female PAG. Staining for NAPE-PLD, FAAH, Iba1, GFAP, and Nissl was performed. Original magnification ×63. C Measurement of relative area of IHC field occupied by NAPE-PLD staining pixels for each sex and region. Female NAPE-PLD immunoreactivity was significantly higher in PAG vs male PAG (*p = 0.02). D Measurement of co-efficient of colocalization for NAPE-PLD with IBa1, Nissl, and GFAP. NAPE-PLD colocalized with Nissl at significantly higher rates in female V1M cortex vs male V1M cortex (****p = 0.001). No significant differences observed in PAG. E Measurement of relative area of IHC field occupied by FAAH staining pixels for each sex and region. FAAH immunoreactivity was significantly higher in female PAG vs male PAG (***p = 0.0042) as well as female PAG vs female V1M cortex (^###p = 0.002). F Measurement of co-efficient of colocalization for FAAH with IBa1, Nissl, and GFAP. No significant differences observed between region or sex. All calculations for co-efficient of colocalization were performed with respect to the relevant enzyme. n = 3/sex for each analysis