Versadoc xl imaging apparatus

Left hemibrains were homogenized and divided into cytosolic and membrane fractions as previously described [50 (link),51 (link)]. For immunoblot analysis, 20 mg of total protein per lane was loaded on 4–12% Bis-Tris sodium dodecyl sulfate polyacrylamaide gel electrophoresis and blotted onto polyvinylidene fluoride membranes. To determine the effects of immunotherapy in levels of Aβ and α-syn, blotted samples from immunized α-syn tg mice were probed with antibodies against Aβ (82E1, 1:1000, IBL), full-length α-syn (1:1000, Syn1, BD), and phosphorylated Tau (pTau) (PHF-1 courtesy of Dr. P. Davies). Incubation with primary antibodies was followed by species-appropriate incubation with secondary antibodies tagged with horseradish peroxidase (1:5000, Santa Cruz Biotechnology, Santa Cruz, CA), visualization with enhanced chemiluminescence, and analysis with a Versadoc XL imaging apparatus (BioRad, Hercules, CA). Analysis of β-actin (Sigma) levels was used as a loading control. The level of human Aβ42 was determined using Human Aβ42 ELISA kit (KHB3441, Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instruction. The cytoplasmic and particulate mouse brain homogenates were diluted in sample dilution buffer (final 50 mg per sample). Optical signals at 450 nm were read on SpectraMax E5 plate reader (Molecular Devices, San Jose, CA).

Frozen samples from the posterior half of the from left mouse hemibrains (including neocortex and hippocampus) were homogenized in detergentcontaining RIPA lysis buffer with phosphatase and protease inhibitors in RIPA lysis buffer (Thermo Scientific; 25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS).
Homogenates were separated into soluble and insoluble fractions through centrifugation at 100,000 g for 60 min. A BCA assay was used to analyze the samples; protein (20 μg/lane) was loaded into each well of a 4–12% SDS-PAGE gel and run in 5% MES (20X) running buffer at 200V for 50 min. The gels were then blotted onto a nitrocellulose membrane, using Thermo Fisher iBlot 2 western detection stacks. Blots were incubated for detection of specific target proteins using the corresponding antibody listed in Table 1 for two days at 4°C. The membrane was then washed and incubated in horseradish-peroxidase-conjugated secondary antibody of the appropriate species (1:1000, Santa Cruz Biotechnology) for 60 min at room temperature. Bands were then washed and visualized by enhanced chemiluminescence (PerkinElmer, Boston, MA), analyzed with a quantitative Versadoc XL imaging apparatus (BioRad, Hercules, CA), and analyzed with Quantity One Software. β-Actin (1:3000, Sigma Aldrich) was used as a loading control.