Odyssey p140 clx infrared imaging system

Mouse brain tissue was homogenized in lysis buffer containing 50 mM Tris-HCL (pH 7.4), 100 mM NaF, 2 mM EDTA, 10 mM β-mercaptoethanol, 2 mM NaVanadate, 8.5% sucrose, 5 μg/ml aprotinin, 100 μg/ml leupeptin, and 5 μg/ml pepstatin. Protein concentrations were determined by using BCA Kits according to the manufacturer’s protocol. Quantitative homogenates were added 5 × sodium dodecyl sulfate (SDS) to heat for 10 min at 99 °C and resolved in 10% SDS-PAGE for separation. After separated, samples were transferred onto nitrocellulose membranes (GE Healthcare, Wauwatosa, WI), and the membranes were blocked with 5% non-fat milk for 1 h at room temperature. Then, the membranes were incubated with primary antibodies including Tau5 (1:1000), Tau1 (1:1000), the anti-phospho-tau antibodies pS199 (1:1000), pT205 (1:1000), pS396 (1:1000), pS404 (1:1000), anti-β-Amyloid (6E10, 1:2000), anti-APP (1:20000), anti-p47^phox (1:250), anti-p67^phox (1:1000), anti-NOX2/gp91^phox (1:1000), anti-GFAP (1:1000), anti-GAPDH (1:20000), and anti-β-Actin (1:20000), followed by the respective IRDye® 800CW or IMDye® 800CW secondary antibodies. The membranes were scanned using an Odyssey P140-CLx Infrared Imaging System (LI-COR, Inc.). Densitometric quantification of protein bands was analyzed using the ImageJ software (National Institutes of Health, Bethesda, MD).

Mouse brain tissue was homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 100 mM NaF, 2 mM EDTA, 10 mM β-mercaptoethanol, 2 mM NaVanadate, 8.5% sucrose, 5 μg/mL aprotinin, 100 μg/mL leupeptin, and 5 μg/mL pepstatin. Protein concentrations were determined using a BCA Kit according to the manufacturer’s protocol. Then, the tissue homogenates were heated in 5× sodium dodecyl sulfate (SDS)-PAGE loading buffer at 99 °C for 10 min. Tissue homogenates were separated on 10% SDS-PAGE, and separated samples were transferred onto nitrocellulose membranes (Whatman Protran®, Sigma). The membranes were blocked with 5% non-fat milk for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies including anti-GFAP (1:1000) and anti-GAPDH (1:1000), followed with corresponding secondary antibodies IRDye® 800CW or IMDye® 800CW antibodies for 1 h at room temperature. The membranes were scanned using the 800-nm channel of an Odyssey® P140-CLx Infrared Imaging System (LI-COR, Inc.). Densitometric quantification of protein bands was analyzed using the ImageJ software (National Institute of Mental Health, Bethesda, MD).

The mouse brain tissues were homogenized in lysis buffer including 50 mM Tris-HCL (pH 7.4), 100 mM NaF, 2 mM EDTA, 10 mM β-mercaptoethanol, 2 mM NaVanadate, 8.5% sucrose, 5 μg/mL aprotinin, 100 μg/mL leupeptin, and 5 μg/mL pepstatin. The protein concentrations were detected using BCA Kits according to the manufacturer’s protocol. The quantitative homogenates were added to a 5× sodium dodecyl sulfate (SDS)-PAGE loading buffer, heated for 10 min at 99 °C, and separated onto 10% or 12% SDS-PAGE. After separation, the samples were transferred onto nitrocellulose membranes (GE Healthcare, Wauwatosa, WI, USA), and the membranes were blocked with 5% non-fat milk for 1 h at room temperature. Then, the membranes were incubated with primary antibodies including anti-Iba1 (1:1000), anti-chemerin (1:1000), anti-CMKLR1 (1:1000), and anti-GAPDH (1:20,000), followed by IRDye^® 800CW or IMDye^® 800CW secondary antibodies. The membranes were scanned using an Odyssey P140-CLx Infrared Imaging System (LI-COR, Inc., Lincoln, NE, USA). Densitometric quantification of protein bands was analyzed by the ImageJ software (National Institutes of Health, Bethesda, MD, USA).