2 μl/min). The stereotaxic coordinates for injection of the immunotoxin solution or the vehicle were AP = −0.2 mm, ML = 1.0 mm BMS-777607 manufacturer and DV = 2.7 mm from bregma according to Franklin and Paxinos [29]. Four months after immunotoxin injections, the survival rate was about 70%

and 85% for animals immunolesioned at 12 and 3 months of age, respectively. Six non-injected 12-month-old 3xTg mice (for analysis of neuropathological alterations at injection time) and all further animals to be analysed solely immunohistochemically were perfused with 4% paraformaldehyde and 0.1% glutaraldehyde in phosphate-buffered saline. This part of the study comprised 16-month-old mice: immunotoxin-treated (3xTg: n = 28; WT: n = 7), sham-injected (3xTg: n = 8; WT: n = 4) and naive (3xTg: n = 20; WT: n = 8), and 7-month-old mice: immunotoxin-treated (3xTg: n = 8; WT: n = 7), sham-injected (3xTg: n = 3; WT: n = 5) and naive (3xTg and WT: n = 6

each). Furthermore, immersion-fixed forebrains from 20 naive, 28 immunolesioned and 6 sham-injected animals were applied to immunohistochemical analyses of cholinergic markers. All fixed tissue samples were primarily cryo-protected by equilibration with 30% phosphate-buffered sucrose. Subsequently, 30 μm-thick frozen sections were cut with a freezing microtome and collected in 0.1 M Tris-buffered saline, pH 7.4 (TBS) containing sodium azide. For biochemical analyses, 21 hippocampi from 7- and 16-month-old immunolesioned animals and untreated AZD1208 datasheet control mice (usually n = 3–4 per animal group) were utilized. In addition, hippocampi from seven mice had been

prepared 4 months following control injection with rabbit-anti-p75. Immunotoxin-treated animals without verified immunolesion were excluded from further investigation. Murine hippocampi were homogenized in 70 μl of lysis buffer (750 mM NaCl, 50 mM Tris/HCl, 2 mM EDTA, supplemented with one Liothyronine Sodium tablet Complete Mini-Protease Inhibitor (Roche, Mannheim, Germany) and 100 μl Phosphatase Inhibitor Cocktail 3 (Sigma, Taufkirchen, Germany) in 10 ml lysis buffer, pH 7.4) per 10 mg tissue. After centrifugation at 17 000 g at 4°C for 20 min, supernatants were stored as soluble fraction at −80°C until use. Pellets were resuspended via sonification in 2% SDS (including protease and phosphatase inhibitors) and centrifuged again. Supernatants were saved as insoluble fraction at −80°C until use. For Western blotting, 50 μg total protein was loaded per lane of a 4–12% VarioGel (Anamed, Groβ-Bieberau/Rodau, Germany). After electrophoresis, proteins were transferred to nitrocellulose membranes (GE Healthcare, Freiburg, Germany) using a semi-dry transfer protocol. Following transfer, membranes were incubated in Tris-buffered saline (0.1 M Tris, 1.5 M NaCl) including 0.5% Tween-20 (TBST) at room temperature for 20 min and boiled in 0.01 M PBS for 5 min for antigen retrieval.