Anti aβ1 42

To evaluate the effects of fermented dairy products on Alzheimer’s disease, 3-month-old transgenic and wild-type female mice were fed a diet with or without fermented sample (2% w/w) for three months (n = 11 mice in each group). At the age of 6 months, the mice were euthanized and the brains were removed. The left hemisphere of the brain was homogenated in RIPA buffer (Wako) with a multi-beads shocker (Yasui Kikai, Osaka, Japan). After centrifugation at 500,000 × g for 20 min, the total protein concentration of the supernatant was measured with a BCA protein assay kit (ThermoScientific, Yokohama, Japan). To quantify Aβ_1–42 (Wako), MIP-1α (R&D systems, MN, USA), TNF-α (eBiosciences, CA, USA), IL-1β (eBiosciences), synaptophysin (Life Science Inc., FL, USA), BDNF (Promega, WI, USA), GDNF (Promega), and NGF (Promega) in the hippocampus, an appropriate ELISA kit was used. The right hemispheres were fixed in 10% formalin (Wako) and analyzed immunohistochemically using the following specific antibodies: anti-Aβ1–42 (polyclonal, Invitrogen, CA, USA), anti-Iba-1 (polyclonal, Wako), and anti-MIP-α (polyclonal, R&D systems).

Cryoprotected hemibrains were serially cut at 50 μm on a freezing microtome and were immunostained with specific antibodies, as described^{69 (link)}. Anti- Aβ1–42 (Invitrogen #700254, 1:1000) was used to define Aβ deposits and anti-Iba1 was used to stain microglia (Wako Chemicals #019-19741; 1:5000). In brief, free-floating sections were incubated overnight in primary antibody followed by PBS (Phosphate buffered saline) washes and incubation in peroxidase-conjugated secondary antibody followed by development using 3′-diaminobenzidine tetrahydrochloride (DAB) as a chromagen. Three serial sections per subject, 0.5 mm apart from each other were quantified in a blinded manner. Quantification of Aβ and Iba1 immunostaining was performed by densitometry using the threshold function on NIH Image J.