Meso scale discovery aβ triplex immunoassay with 4g8 detection

Hippocampal homogenates (same as the IB samples) from 9-month-old WT-SynRFP (n = 4), PSAPP-SynRFP (n = 3), and PSAPP-SynCav1 (n = 3) were tested using the Meso Scale Discovery Aβ triplex immunoassay with 4G8 detection (catalog # K15141E-1, MSD Rockville, MD, USA) to quantitate levels of Aβ42, Aβ40, and Aβ38. The kit was run according to the manufacturer’s instructions. Briefly, 25 μL of SULFO-TAG conjugated 4G8 detection antibody and 25 μL of WT sample or 3 μL of AD samples were added to a blocked MSD multiplex plate pre-coated with capture antibodies specific for Aβ42, Aβ40, and Aβ38 and incubated for 2 h with shaking at RT. The plate was read on an MSD SECTOR Imager 2400 after addition of read buffer. Concentrations of Aβ42, Aβ40, and Aβ38 in samples were backfitted from the respective standard curves using MSD Workbench software. Total Abeta was quantified in the AD brain samples using Meso Scale Discovery’s Electrochemiluminescence R-plex assay (MSD, USA). The assay was performed following the manufacturer’s instruction using MSD GOLD 96-well small spot streptavidin plate and recommended diluents. Samples were tested at 1:8 dilution. Plates were analyzed on an MSD SECTOR Imager 2400 instrument. All data was normalized by protein concentration measure by Bradford assay. 3–4 mice per group was used to get quantifiable data.