(i) Double co-localization of caspase-1 and GFAP in cortical astrocytes*: cortical brain sections were defrosted for 30 min and incubated with 0.25% Triton in PBS solution for 10 min. Sections were blocked for 1 h/RT with 10% normal goat serum in TBS/T (0.1%) and were incubated overnight at 4°C with the following primary antibodies; anti-caspase-1 (1/50, Santa Cruz) 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. Negative controls were performed by replacing the respective primary antibodies with IgG isotype control and concentration-matched irrelevant antibodies (Figure 1F). Sections were mounted onto glass slides with Dako Fluorescent Mounting medium (Dako North America Inc., CA, USA). All the images were analyzed with the ImageJ 1.42 software (NIH). We determined fluorescence intensity of three different animals per group by randomly taking 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
