Human rat β amyloid 42 elisa kit

Blood was collected from mice under a fed condition. The insulin concentrations in the sera were determined by ELISA (Mercodia Ultrasensitive Mouse Insulin ELISA, Mercodia AB). The serum concentrations of β-amyloid 40 and 42 were quantified using a Human/Rat β-Amyloid (40) ELISA kit (WAKO) and Human/Rat β-Amyloid (42) ELISA kit (WAKO).

Mouse brains were dissected in 1X phosphate‐buffered saline (PBS) divided into halves. Hippocampi and cortex sections were extracted and snap‐frozen. Frozen cortex sections were homogenized in RIPA buffer (10% sodium deoxycholate, 10% SDS, 0.5M EDTA, protease inhibitor cocktail in 1X PBS). Samples were then diluted 1:10 in 5M guanidine‐HCl and allowed to rotate overnight at room temperature. Samples were diluted 1:10 again in a Tris buffer solution (50 mM Tris–HCl pH = 8, 0.03% Tween‐10, protease inhibitor cocktail) and centrifuged at 16,000 g for 20 min at 4°C. Supernatants were collected and diluted (1:20) for Aβ analysis. Aβ levels were measured using a Human/Rat β‐amyloid (42) ELISA Kit (Wako Chemicals, Richmond VA, USA). The ELISA experiments were performed as per manufacturer's instructions. Protein levels were measured via Bradford Protein Assay Kit (Pierce) and used to normalize Aβ levels measured.

Frozen tissues of the retina of OXYS rats and Wistar rats and for evaluation of effects of the SkQ1 (n = 5) were homogenized in lysis (RIPA, radioimmunoprecipitation assay) buffer (50 mmol/L Tris-HCl, pH 7.4; 150 mmol/L NaCl; 1% of Triton X-100; 1% of sodium deoxycholate; 0.1% of SDS; and 1 mmol/L EDTA) supplemented with a protease inhibitor cocktail (P8340; Sigma-Aldrich, St. Louis, MO, USA). After incubation for 20 min on ice, the samples were centrifuged at 9660× g at 4 °C for 30 min, and the supernatants were transferred to new tubes. Total protein was quantified with the Bradford assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Samples were resolved by SDS-PAGE on 12% gels in Tris-glycine buffer (25 mmol/L Tris base, 190 mmol/L glycine, and 0.1% of SDS) and transferred to nitrocellulose membranes. The membranes were probed with specific antibodies against S6 ribosomal protein and phospho-S6 ribosomal protein (p-S6) (1:1000; Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight with an anti-actin antibody (cat. # ab1801; 1:1000; Abcam, Cambridge, MA, USA). Signals were scanned and the intensity of the bands was measured in ImageJ, version 1.41 (NIH, Bethesda, MD, USA).
The Human/rat β Amyloid (42) ELISA Kit (Wako, Japan) was employed to measure Aβ levels in the retina of all the rat groups according to the manufacturer’s instructions.

APP_NL-H4 cells were plated at a density of 500,000 cells/well in 6-well plates. After resting for 24 h, the cells were treated with 1 ppm methanol extract of loquat leaves, UA or Amygdalin for 24 h, and the medium was replaced with the same medium containing the extract or amygdalin. The cells were incubated for 24 h and the medium was harvested. Aβ total of 42 levels in the medium from the APP_NL-H4 cells were measured using ELISA kits (IBL, Ltd., Fujioka, Japan).
Mouse cortices were homogenized with a tissue homogenizer (Rotor: TLA45, Beckman Coulter) on ice in Tris-buffered saline (TBS) (25 mM Tris–HCl pH 7.4, 137 mM NaCl, 2.68 mM KCl) containing a protease inhibitor cocktail (Roche Diagnostics Ltd, Mannheim, Germany) and phosphatase inhibitor cocktail (Nacalai Tesque). The homogenates were ultracentrifuged at 45,000×g for 20 min at 4 °C. The cortical Aβ42 levels were determined using a Human/Rat β amyloid (42) ELISA Kit (Wako, Tokyo, Japan). The results were measured using a Benchmark Microplate Reader (Bio-Rad, Hercules, CA, USA).