Anti synaptophysin

For immunofluorescence, tissues were permeabilized in 0.1% PBS-T for 15 min. To retrieve antigens, the samples were boiled in 0.1 M citrate buffered saline (pH 6.0) for 5 min. The sections were cooled for 30 min and then washed twice in PBS (5 min each). After washing in PBS-T for 30 min, the sections were blocked for 1 h in blocking solution (4% normal donkey serum in PBS-T). The sections were incubated with primary antibody overnight at 4 °C. Anti-oligomer A11 (Invitrogen, CA, USA) (1:100), Anti-6E10 (Aβ_1–16) (Covance, NJ, USA) (1:500), Anti- D54D2 (Aβ_1–40, Aβ_1–42) (Cell Signaling, MA, USA) (1:500), anti-synaptophysin (Agilent Dako, CA, USA) (1:250), anti-TH (Santa Cruz Biotech, TX, USA) (1:250), and anti-Ki67 (Cell Signaling, MA, USA) (1:250) antibodies were used. Alexa 488 and Cy3-conjugated secondary antibodies (Jackson Laboratory, Bar Harbor, ME, USA) were used. The sections were counter-stained and mounted using VECTASHIELD mounting medium with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, CA, USA). The images were visualized and photographed by confocal fluorescence microscopy (Carl Zeiss, Thornwood, NY, USA).

In brief, the tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and processed by standard histological methods. From each selected paraffin block, 4 μm serial sections were cut. Immunohistochemical (IHC) studies were performed with avidin-biotin-peroxidase complex kits according to the manufacturer’s instructions (Agilent, Dako, CA, USA). Primary antibodies (anti-EphB2 [rabbit polyclonal, dilution 1:200, R&D Systems, MN, USA AF467], anti-β-catenin [rabbit polyclonal, dilution 1:250, Sigma-Aldrich, Merk, Germany c2206], anti-LYZ [rabbit polyclonal, dilution 1:5000, Agilent, Dako, CA, USA. A0099], anti-c-kit [rabbit polyclonal, dilution 1:200, Santa Cruz Biotechnology, Heidelberg, Germany sc-5535], anti-synaptophysin [rabbit polyclonal, dilution 1:200, Agilent, Dako, CA, USA. A0010] and anti-CD31 [rabbit polyclonal, dilution 1:200]) were incubated in a humidified chamber at 4 °C overnight. After that, the samples were developed with liquid DAB + substrate chromogen system (Agilent, Dako, CA, USA) under microscope (DM3000, Leica, Wetzlar, Germany) and the results were counted by two independent researchers. The method of IHC READING calculation was as follows: Staining intensity: 0 = no staining, 1 = weak staining, 2 = moderate staining, 3 = strong staining; percentage of positive cells: 0 = 0%, 1 = 1–10%, 2 = 10–50%, 3 ≥ 50%.

Cell lysis and immunoblotting was essentially performed as described previously [42 (link)]. In brief, cells were washed once with ice-cold PBS and lysed with PhosphoSafe lysis buffer (Merck Millipore, Darmstadt, Germany). Following clearance of the lysates by centrifugation, their total protein concentrations were determined with the DC Protein Assay (BioRad). Equal amounts of proteins were fractionated by PAGE on mini-PROTEAN TGX any-kD precast gels, or TGX Stain-Free FastCast gels (BioRad) and blotted to PVDF membranes. Membranes were blocked with nonfat dry milk or bovine serum albumin and incubated with primary antibodies (CgA: Monoclonal Mouse Anti-Human Chromogranin A, clone DAK-A3, Dako, Glostrup, Denmark; SYP: Anti-Synaptophysin, Dako; SSTR2: Anti-Somatostatin Receptor 2 antibody [UMB1]-C-terminal #ab134152, Abcam, Cambridge, UK) overnight at 4 °C. After washing and incubation with horseradish peroxidase-linked secondary antibodies, chemoluminescent detection of proteins was done on a ChemiDoc XRS imaging system (BioRad) with Amersham ECL Prime Detection Reagent (GE Healthcare, Munich, Germany). The signals for the proteins of interest were normalized to those for the housekeeping genes GAPDH or HSP90.