For microglial analysis, z-stack images were analyzed with ImageJ [National Institutes of Health (NIH)]. Three images were taken from the coronal sections of mPFC of each mouse stained with anti-Iba1 and anti–TNF-α (n = 8 mice per group). Microglia activation status was categorized into ramified, intermediate, amoeboid, or round on the basis of cell morphology from Iba1 staining. Then, the activation status was categorized as either activated (intermediate, amoeboid, and round) or surveying (ramified) as previously described (58). The quantity and percentage of microglia in each category as well as “activated” microglia were reported per animal. Percentages of TNF-α–positive microglia per total microglia were also calculated with 20× z-stack images (n = 7 mice for control shRNA and n = 8 mice for Gstm1 shRNA).

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