We performed sandwich ELISA using Human βAmyloid (1–42) ELISA Kit or Human βAmyloid (1–40) ELISA Kit (298-62401 and 292-62301, FUJIFILM Wako PureChemical Corp., Osaka, Japan). Total proteins extracted from 20 mg of mouse cortex tissues by 1 mL of RIPA buffer (10 mM Tris-HCl pH7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate) were ultra-centrifuged at 100,000 × g at 4 °C for 1 hour. The supernatants were diluted to 100 µL and applied to Aβ1–42 (extract from 100 µg cortex/well) and Aβ1–40 (extract from 500 µg cortex/well) ELISA plates and measured following the manufacturer’s instructions.
The ELISA plates were incubated at 4 °C overnight, washed five times with 1x wash solution, added with 100 µL of HRP-conjugated antibody solution, and incubated at 4 °C for 1 or 2 h in Aβ1–42 or Aβ1–40 ELISA kits, respectively. After washed five times by 1x wash solution, the ELISA plates were added with 100 µL of TMB solution, incubated at room temperature for 30 min, and added with 100 µL of stop solution. Absorbance was measured at 450 nm using a plate reader (SPARK 10 M, TECAN, Grodig, Austria).
The ELISA plates were incubated at 4 °C overnight, washed five times with 1x wash solution, added with 100 µL of HRP-conjugated antibody solution, and incubated at 4 °C for 1 or 2 h in Aβ1–42 or Aβ1–40 ELISA kits, respectively. After washed five times by 1x wash solution, the ELISA plates were added with 100 µL of TMB solution, incubated at room temperature for 30 min, and added with 100 µL of stop solution. Absorbance was measured at 450 nm using a plate reader (SPARK 10 M, TECAN, Grodig, Austria).