Ki 67 clone d3b5

Hematoxylin and eosin (H&E) staining and immunohistochemistry were performed using 4 μm thick formalin-fixed, paraffin-embedded tissue sections. Briefly, slides were soaked in xylene, passed through graded alcohols and put in distilled water. Slides were then pre-treated with 1.0-mM EDTA, pH 8.0 or 1.0 mM Citrate (Zymed) in a steam pressure cooker (Decloaking Chamber, BioCare Medical) as per manufacturer’s instructions, followed by washing in distilled water. All subsequent steps were performed at room temperature in a hydrated chamber. Slides were pre-treated with Peroxidase Block (DAKO) for 5 minutes to quench endogenous peroxidase activity, followed by Serum free Protein Block (DAKO) for 20 minutes. Rabbit monoclonal antibody against mouse KI-67 (clone D3B5, Cell Signaling Technology) was applied and slides were incubated overnight at 4 °C. Slides were washed 2X in 50 mM Tris-Cl, pH 7.4 then followed with goat anti-rabbit IgG-HRP (SouthernBiotech), and incubated for 15 minutes. After further washing, immunoperoxidase staining was developed using a DAB chromogen (DAKO) and counterstained with hematoxylin. Images were acquired with the Nikon Eclipse TE2000 and were captured with the Hamamatsu Orca ER digital CCD camera and analyzed with NIS elements 4.13 software. Images were shown as 100X (10X ocular and 10X objective lens) and 400X (10X ocular and 40X objective lens).

Mice were transcardially perfused with PBS containing 5 IU/ml heparin (De Pannemaeker, Ghent, Belgium), followed by perfusion with 4% paraformaldehyde. Brain and spinal cord tissue were dissected, dehydrated, and embedded in paraffin blocks. Alternatively, for vibratome sections brains were stored in low-melting agarose until further processing. Sections of 5 µm were stained with Luxol fast blue (Solvent Blue 38, practical grade; Sigma Genosys) for assessment of demyelination, or incubated with antibodies against CD3 (clone CD3–12; Serotex), Mac-3 (clone CD107b, M3/84; Becton Dickinson Biosciences), B220 (clone RA3-6B2; Becton Dickinson Biosciences), amyloid precursor protein (APP; clone 22C11; Millipore), Iba-1 (Wako Chemicals), or Ki67 (clone D3B5; Cell Signaling). Sections were rehydrated and incubated in antigen retrieval buffer (Dakopatts). Endogenous peroxidase activity was blocked by immersing slides in 3% H₂O₂. Nonspecific binding was blocked by incubating sections in 5% NGS and 0.1% Triton X-100. Primary antibodies were incubated overnight at 4 °C.