We also appreciate the support and assistance of the Division of Laboratory Animal Resources staff. [11C]PK11195 PET scans acquired before and after immunization, and by post-mortem immunohistochemical and real-time PCR evaluation. A oligomer composition was assessed by immunoblot analysis in the frontal cortex of aged immunized and non-immunized control animals. Results All juvenile animals developed a strong and sustained serum anti-A IgG antibody response, whereas only 80?% of aged animals developed detectable antibodies. The immune response in aged monkeys was more delayed and significantly weaker, and was also more variable between animals. Pre- and post-immunization [11C]PK11195 PET scans showed no evidence of vaccine-related microglial activation. Post-mortem brain tissue analysis indicated a low overall amyloid burden, but revealed a significant shift in oligomer size with an increase in the dimer:pentamer ratio in aged immunized animals compared with non-immunized controls (and 4C. Supernatants were transferred into appropriate ultracentrifuge tubes, and the pellets were rehomogenized in 0.3?mL of buffer, then separated again by centrifugation for 10 minutes at 1000?and 4C. The second supernatant was collected and combined with the first supernatant and separated by centrifugation DNA2 inhibitor C5 for 1 hour at 100,000?and 4C. This final supernatant was collected to serve as the cytosolic fraction, and the remaining pellet was resuspended in 0.2?mL of buffer and rehomogenized to give the membrane fraction. The bicinchoninic acid protein assay was used to determine the protein concentration of the samples. For immunoblot analysis, samples (20?g) were loaded onto a 4 to 12% Bis-Tris gel (Invitrogen, Carlsbad, CA, USA) and run with MESCSDS running buffer (NuPAGE; Invitrogen) at 200?V for 45 minutes on ice. Gels were transferred to a 0.2?m nitrocellulose membrane, in transfer buffer (1?l Tris-glycine buffer containing 20% methanol) at 400?mA for 1.5 hours. Membranes were boiled in phosphate-buffered saline for 5 minutes, blocked with 3% BSA in Tris-buffered saline and 0.05% Tween 20 (TBS-T) for 1 hour, and incubated overnight at 4C with anti-A antibody (6E10, Signet Laboratories, Dedham, MA, USA). Membranes were washed in TBS-T for 1 hour, and incubated in secondary antibody for 1 hour at room temperature. Membranes were washed for 30 minutes in TBS-T and then for five minutes in TBS alone. Detection was carried out with a commercial reagent (Western Lightning Chemiluminescence Reagent Plus; PerkinElmer, Waltham, DNA2 inhibitor C5 MA, USA) and visualized by enhanced chemiluminescence. Membranes were analyzed with a Versadoc XL imaging apparatus (BioRad, Hercules, CA, USA). Analysis of actin levels (C4, Millipore, Temecular, CA, USA) was used as a loading control. A42 concentrations were also measured by ELISA in accordance with the manufacturers protocol (Invitrogen, Camarillo, CA, USA). Histopathologic and immunohistochemical evaluation Animals were killed and perfused with saline, the brains were then removed immediately and processed for neuropathologic analysis as described previously [34]. Briefly, brains were bisected sagittally, then the left half was fixed in 10% formalin and tissue blocks were embedded in paraffin wax, while the right half was micro-dissected and snap-frozen at ?80C. Sections of brain were stained with hematoxylin and eosin, or immunostained for the macrophageCmicroglia-associated protein Iba-1 (1:500, Wako Chemicals USA, Richmond, VA, USA) and the Mouse monoclonal to IFN-gamma T-cell marker CD3 (polyclonal, 1:500, Dako, Carpenteria, CA, USA). The presence of amyloid pathology was evaluated by Bielschowsky silver stain and immunohistochemical stains for A (clone 6F/3D, 1:100; Dako (formic acid pre-treatment)) and total tau (polyclonal antibody, 1:200; Dako). To assess if vaccination-induced anti-A antibodies bind to A plaques, colabeling for A and anti-monkey IgG (1:500, Rockland, Gilbertsville, PA) was performed. Prussian blue iron stain was used to screen for microhemorrhages. Slides were dewaxed, rehydrated, and immersed in 10% potassium ferrocyanide for 5 minutes, followed by immersion in equal parts of 20% hydrochloric acid and 10% potassium ferrocyanide for 30 minutes. Slides were counterstained with Nuclear fast red. Quantitative image analysis Amyloid plaque burden, vascular amyloid, and. DNA2 inhibitor C5
