30 μm-thick coronal brain sections were permeabilized with 0.3% Triton-X and incubated with NH4Cl 50 mM for 30 min at room temperature. Blocking was performed with a solution of 2% bovine serum albumin and 3% fetal bovine serum for 2 h at room temperature. Afterward, sections were incubated overnight at 4°C with primary antibodies [rabbit polyclonal anti-IBA1 (1:1,000, Wako) and goat polyclonal anti-IL1β (1:500, R&D Systems)]. After washing, secondary antibodies [1:500 anti-rabbit Alexa Fluor 555, anti-goat Alexa Fluor 405 (Invitrogen)] were incubated for 1 h at room temperature. The sections were mounted with the Mowiol mounting medium.
For IBA1/IL1β quantification, 1024 × 1024 pixel confocal fluorescent image stacks from these brain sections were obtained with a TCS SP5 LEICA confocal microscope, using an X20 objective. We obtained pictures of the CA1 hippocampus and motor cortex. ImageJ software (https://imagej.nih.gov/ij/ ) was used for image quantification. N = 5–7 animals per group were analyzed, and three brain slices were analyzed per animal.
For IBA1/IL1β quantification, 1024 × 1024 pixel confocal fluorescent image stacks from these brain sections were obtained with a TCS SP5 LEICA confocal microscope, using an X20 objective. We obtained pictures of the CA1 hippocampus and motor cortex. ImageJ software (