four medial-frontal-cortex sections for each one. At least 800–1000 cells were counted per experimental condition. (ii) Double labeling of NLRP3-inflammasome and GFAP in cultured astrocytes*. Treated/untreated astroglial cells were previously incubated with 0.25% Triton in PBS solution for 10 min for cellular permeabilization. Cells were then blocked for 1 h at room temperature with 10% normal goat serum in TBS/T (0.1%), and were incubated overnight at 4°C with the following primary antibodies; anti-NLRP3 (1/150, AdipoGen) and anti-GFAP (1/400, Sigma–Aldrich), following incubation with the respective Alexa-fluor-conjugated secondary antibodies (1/500, Invitrogen, Molecular Probes). Nuclei were stained with DAPI. All the negative controls were also performed as described above (i). Cells were mounted onto glass slides with Dako Fluorescent Mounting medium (Dako North America Inc., CA, USA), and confocal images were analyzed with the ImageJ 1.42 software (NIH). All the confocal images were acquired using the same settings, while fluorescence distribution was acquired with a Leica TCS-SP2-AOBA confocal laser-scanning microscope (Leica Microsystems, Heidelberg GmbH, Mannheim, Germany) using the 10× Plan-HCX PL APO CS10 and the 40× Plan-HCX PL APO CS40 × 1.25 oil objectives. All the confocal images were acquired with the same settings and the fluorescence distributions were analyzed by the Leica
