Anti ki67

Paraffin-embedded sections were firstly deparaffinized and then incubated with rabbit polyclonal anti-Ki-67 (#12075, Cell Signaling Technology) or rabbit polyclonal anti-CNN1 (#17819, Cell Signaling Technology) primary antibody at 4°C overnight. After being washed by TBST three times, the sections were then incubated with horseradish peroxidase–conjugated goat anti-mouse antibody (#4414, Cell Signaling Technology). The sections were washed by TBST three times and the signal was tested utilizing DAB Substrate Kit. Images were gained utilizing a microscopy.

Bone marrow cells from untreated animals were collected as described above. First, we measured the expression of FPR2 in LSK cells. The anti-FPR2-FITC antibody was used (Bioss, Massachusetts, USA) to quantify this expression by flow cytometry. Afterwards, bone marrow cells were treated with rAnxA1 (100 nM) for 12 h, and viability of the LSK population determined using 7-aminoactinomycinD (7-AAD; Sigma Aldrich, USA); cell cycle phases and expression of Ki67 and Notch-1 were also evaluated. These quantifications were carried out by flow cytometry. Cells were fixed in 2% paraformaldehyde for 30 min, washed with 0.1 M glycine, and permeabilized with 0.001% Triton X-100. Subsequently, 2 × 10⁶ cells were incubated with primary anti-cyclin B1, anti-Notch-1 or anti-Ki67 (Cell Signaling Technology) antibodies for 2 h, and then 40 min with secondary rabbit Anti-IgG Alexa Fluor 488 (Molecular Probes/Invitrogen, USA). Analyses were performed on the LSK population gated as described above, using an Accuri C6 flow cytometer (Becton Dickinson, USA) and the FlowJo software.

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).

Cells were placed on glass coverslips in 12-well plates and fixed with absolute methanol at −20 °C for 5 min. Spheres were prepared as described above. Cells and spheres were blocked with 0.5% (v/v) Triton X-100 in PBS and 3% (w/v) bovine serum albumin (BSA) and incubated with anti-Acetyl-Histone H3 (Lys9) (Cell Signaling, Danvers, MA, USA), anti-BMI-1 (Millipore, Billerica, MA, USA), and anti-Ki-67 (Cell Signaling, Danvers, MA, USA) as indicated. Cells were then washed three times and incubated with Fluorescein isothiocyanate (FITC) or Tetramethylrhodamine (TRITC)-conjugated secondary antibody for 60 min at RT and stained with Hoechst 33342 for visualization of DNA content. Images were captured using a QImaging ExiAqua monochrome digital camera attached to a Nikon Eclipse 80i Microscope (Nikon, Melville, NY, USA) and visualized with QCapturePro software.