Cells were then fixed with 4% PAF GW-572016 in vivo and stained for ED1. Following Bioporter treatment, primary rat monocytes (~ 1 × 106) were incubated in 500 μl of culture medium ± 10 μg rat Aβ1-42 (Calbiochem) at 37 °C/5% CO2. Supernatant was collected at 0.2, 3 and 24 h. Subsequently, supernatants were evaluated for NGF and cytokine secretion by ELISA. To evaluate effective transfection efficiency, following incubation, pmaxGFP transfected cells were washed with PBS and then fixed with 4% PFA for 30 min at 4 °C. Following washes, cells were stained with nuclear DAPI (1:10,000, Sigma) for 20 min. Cells

were then washed with PBS and visualized under the fluorescence microscope (Leica DMIRB). DAPI and GFP microscope images were obtained using Improvision Openlab 4.0.4 imaging software captured with DAPI and FITC filter sets, respectively. Cell viability was determined by analyzing the number of necrotic and apoptotic cells by flow cytometry (BD Accuri C6, BD Biosciences) using annexin V-FITC and propidium iodide (PI; Annexin V-FITC Apoptosis Detection Kit, BD Biosciences) www.selleckchem.com/products/SB-203580.html staining according to manufacturer’s instructions.

Gating was performed on monocytes based on side-scatter and forward-scatter properties. All necessary controls were included. Cholinergic neurons in organotypic brain slices were cultured as previously described (Weis et al., 2001, Humpel and Weis, 2002 and Böttger et al., 2010). Briefly, the basal nucleus of Meynert of postnatal day 10 (P10) rats was dissected under aseptic conditions, 400 μm slices were cut with a tissue chopper (McIlwain, USA), and the slices were placed on a 30-mm Millicell-CM 0.4 μm pore membrane culture plate inserts (7–8 slices per membrane). Slices were cultured in 6-well plates at 37 °C/5% CO2 with 1.2 ml/well of pooled and filtered medium containing pEF-NGF or pEF-(−)-transfected cells or slice medium supplemented with or isothipendyl without 10 ng/ml

recombinant NGF for 2 weeks. We have previously established that 400 μm brain slices become thinner following 2 weeks of incubation with a thickness of approximately 100 μm. This flattening is also an internal control indicating a good preparation and dissection. Slices that did not flatten were immediately removed from the experiments. Immunohistochemistry was performed as previously described (Zassler et al., 2005b, Zassler and Humpel, 2006, Böttger et al., 2010 and Hohsfield and Humpel, 2010). Brain slices were fixed for 3 h at 4 °C in 4% PFA/10 mM PBS, washed in PBS and stored at 4 °C until use. Cultured cells were fixed for 30 min in 4% PFA. After fixation, slices/cells were washed with 0.1% Triton/PBS (T-PBS) for 30 min at 20 °C and then pretreated with 5% methanol/1% H2O2/PBS for 20 min to destroy endogenous peroxidase. Slices/cells were then washed 3 × with PBS and blocked in 20% horse serum/0.2% BSA/T-PBS for 30 min.