Ox 42

After behavior test, animals were intracardially perfused with PBS followed by 4% PFA as previously described^{37 (link),38 (link)}. Briefly, the brain sections were cut and incubated at room temperature in PBS with 0.01% Triton X-100 for 30 min and followed by blocking with 3% bovine serum albumin for 1 h. For immunolabeling, brain slices were probed with primary antibody overnight at 4 °C. Antibodies included phospho-CaMKII (Thr286) (1:200)^{39 (link),40 (link)}, laminin, GFAP, OX-42, and S100β (1:500, Millipore, U.S.A), Nuclei were stained with DAPI (4, 6-diamidino-2- phenylindole) (Sigma-Aldrich, U.S.A). After washing, the sections were incubated with Alexa Fluor 488-conjugated anti-rabbit IgG and Alexa Fluor 594-conjugated anti-mouse IgG (Invitrogen, Carlsbad, CA). Signals were visualized by using a Zeiss LSM 510 confocal microscope. The relative fluorescence intensity of immunostaining was quantified by using Image J software (NIH, Bethesda, MD, USA).

Wistar rats were bred in the INBIOMED animal housing facility, University of Buenos Aires, Buenos Aires, Argentina. Rats were kept in a 12 h light-dark cycle at 22°C ± 1°C with access to food and water ad libitum. 1–2 day-old Wistar rat pups were decapitated and their brains removed and freed from meninges. Cells were mechanically dispersed and seeded in previously poly-L-lysine-coated culture flasks, and held in DMEM/F-12 supplemented with 10% FBS, 50 μg/ml streptomycin and 50U penicillin, at 37°C in 5% CO₂. Medium was replaced twice a week. After 11–14 days, microglial cells were detached from astrocytes by shaking for 1–2 h at 110 rpm, at 33°C in a Thermo Scientific Orbital Shaker (Thermo Fischer Scientific, Germany). Supernatants were collected and centrifuged, and microglia was seeded in uncoated plates in supplemented DMEM containing 10% FBS and 2 mM L-glutamine and left to stabilize overnight at 37°C in a 5% CO₂ atmosphere before adding the drugs in fresh supplemented DMEM containing 2% FBS and 2 mM L-glutamine. Cells were stained with a microglial marker (anti-rat CD11b monoclonal antibody, OX-42, Millipore) to assess their purity, which was routinely close to 98%.

Cells were washed in PBS and fixed in 4% PBS–PFA for 10–15 minutes at RT. For p65 and c-Rel immunocytochemistry cells were incubated in ice cold methanol for 10 minutes at -20°C. For HMGB1 staining slides were incubated in Citrate buffer and subjected to microwave heating at 350 W for 5 minutes, then left to reach RT for 20 minutes and washed in PBS. Slides were incubated in blocking solution containing 10% donkey serum or 10% goat serum (depending on the source of the secondary antibody) in PBS for 1 hour at RT. Subsequently, cells were incubated overnight at 4°C with the following primary antibodies: anti-CD11b 1:100 (OX-42, Millipore), anti-p65 1:30 (BD Biosciences), anti-c-Rel 1:50 (sc-70, Santa Cruz), anti-HMGB1 1:100 (ab79823, Abcam). After rinsing, slides were incubated with the appropriate secondary antibodies for 1 hour at RT. Cells were washed in PBS and mounted in mounting medium Vectashield (Vector Laboratories). Negative control slides were incubated with blocking solution instead of the primary antibodies. Staining was visualized in a fluorescence microscope (Axiophot; Carl Zeiss, Jena, Germany). Quantification of fluorescence intensity was done with ImageJ Software (NIH, USA).