Cytokine Enzyme-Linked Immunosorbent Assay (ELISA)

Commercial kits were used to quantify the concentration of the pro-inflammatory cytokines IL-1β, IL-6, and TNFα in brain and spleen extracts (BioLegend, San Diego, CA). Briefly, sandwich ELISAs were performed in 96-well, flat-bottom MaxiSorp microtiter plates (Nunc, Roskilde, Denmark). Microplates were coated with the capture antibody for 18 h at 4 °C. After washing with PBS-Tween-20 (0.05 %) and blocking for 60 min at room temperature with 2 % PBS-BSA, plates were incubated at room temperature for 2 h with standard or samples, washed three times, and incubated for 1 h with the detection antibody at room temperature.

Bound detection antibodies were identified using 1:1000 diluted Avidin-HRP and TMB as a substrate. Optical density was read before and after the reaction was stopped with H₂SO₄ 2 N at 450 and 630 nm, respectively. Results were expressed in pg/mL per mg of protein in the respective soluble extract.