Multiplex ELISA

The remaining brain hemisphere from each animal was flash frozen in liquid nitrogen and stored at −80°C. Production of AD-related and immune-related factors was assessed by multiplex ELISA (Eve Technologies, Calgary, Canada). Brain tissue was harvested after Morris water maze testing, thawed on ice and lysed in RIPA buffer (catalog # 89900, Thermo Fisher Scientific) containing a protease inhibitor cocktail (catalog # 11697498001, Roche Diagnostics, Basel, Switzerland). Lysates were collected, briefly sonicated, brought to a volume of 1 mL and centrifuged at 13,000 rpm for 20 min at 4°C. Supernatants were collected, diluted ten-fold in RIPA buffer and protein concentration was measured and normalized to 400 μg/mL. Samples were flash frozen in liquid nitrogen and shipped on dry ice to Eve Technologies for analysis. Discovery Assay protein arrays and Custom-Plex Assays were used to quantify the following analytes: Human Aβ and Tau 2-Plex Assay (custom panel: Aβ42, Aβ40), Human Supplemental Biomarker 10-Plex Discovery Assay [catalog # HDHSB10: neural cell adhesion molecule (NCAM), soluble vascular cell adhesion molecule 1 (sVCAM-1), soluble intercellular adhesion molecule 1 (sICAM-1), platelet-derived growth factor (PDGF-AA, PDGF-AB), cathepsin D], mouse matrix metalloproteinase (MMP) 5-Plex Discovery Assay (catalog # MDMMP-S,P: MMP-2, MMP-3, MMP-8, MMP-9, MMP- 12), mouse transforming growth factor beta (TGF-β) 3-Plex Discovery Assay (catalog # TGFβ1-3: TGF-β1, TGF-β2, TGF-β3), and Mouse Focused 10-Plex Discovery Assay [catalog # MDF10: interleukins (IL-1β, IL-2, IL-6, IL-10, IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemoattractant protein 1 (MCP-1), and tumor necrosis factor alpha (TNF-α)].