Protein Quantification in Brain Tissue

For protein determination, one series of brain tissue was sonicated in cold phosphate-buffered saline (PBS) (Sigma life science, St. Louis, MO, USA) containing phosphatase inhibitors (Sigma-Aldrich, Soeborg, DK) and cOmplete mini, EDTA-free proteinase inhibitor cocktail (Roche, Basel, Switzerland). The total protein content was measured using the bicinchoninic acid (BCA) assay according to the manufacturer’s protocol (P4417-100TAB, Thermo Scientific, Waltham, MA, USA).
Electrochemiluminescence analysis was performed on brain, serum, cells, and media using the MSD Mouse Proinflammatory V-Plex Plus Kit (IFNγ, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, CXCL1, TNF; K15012C, Mesoscale Discovery), the U-Plex Kit (TNFR1; K15069L, Mesoscale), and the R-Plex kit (TNFR2; K150ZSR-2, Mesoscale Discovery) according to the manufacturer’s instructions. Plates were read using the MSD QuickPlex (SQ120) Plate Reader (Mesoscale Discovery), and the data analyzed using the MSD Discovery Workbench software as detailed [20 (link)].
IL-1Ra ELISA was performed as specified in the Quantikine mouse IL-1Ra assay (MRA00, R&D). TLR2 ELISA was performed as specified in the mouse TLR2 Elisa kit (ab224880, Abcam). The measured protein was normalized to the total protein content measured using the BCA assay [32 (link)].