Rabbit polyclonal anti aif 1 antibody

One out of every eight free-floating sections from the hippocampal formation was incubated with one of the following primary antibodies: rabbit polyclonal anti-AIF-1 antibody (1:300; Thermo Fisher), mouse monoclonal anti-NeuN antibody (1:250; Merck Millipore), rabbit polyclonal anti-GFAP antibody (1:400; Abcam), mouse monoclonal anti-GAD67 antibody (1:500; Merck Millipore), or rabbit monoclonal Ki-67 antibody (1:100; Thermo Scientific). Sections were then incubated for 2 h at room temperature with a Cy3-conjugated anti-rabbit secondary antibody (1:200, Jackson ImmunoResearch Laboratories, Inc.) for AIF-1, GFAP, and Ki-67 immunohistochemistry, and with a Cy3-conjugated anti-mouse secondary antibody (1:200, Jackson ImmunoResearch Laboratories, Inc.) for GAD67 and NeuN immunohistochemistry. Nuclei were counterstained with Hoechst-33342 (Sigma-Aldrich).

One out of every 8 serial brain sections from the hippocampal formation was incubated with one of the following primary antibodies: mouse monoclonal anti-NeuN antibody (1:250; Merk Millipore, Burlington, MA, USA) or rabbit polyclonal anti-AIF-1 antibody (1:300; Thermo Fisher Scientific, Waltham, MA, USA). Sections were then incubated for 2 h at room temperature with a Cy3-conjugated anti-mouse secondary antibody (1:200, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) or with a Cy3-conjugated anti-rabbit secondary antibody (1:200, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). Nuclei were counterstained with Hoechst-33342 (Sigma-Aldrich, St. Louis, MO, USA). Fluorescent images were acquired using a Nikon Eclipse TE600 microscope equipped with a Nikon Digital Camera DXM1200 ATI System (Nikon Instruments Inc., Melville, NY, USA). The number of Hoechst-positive and NeuN-positive cells were manually counted using the Image Pro Plus software (Media Cybernetics, Silver Spring, MD, USA) and established as cells/mm³. Starting from 20X magnification images of AIF-1-stained hippocampal slices, AIF-1 positive microglial cell body size was manually drawn using the Image Pro Plus measurement function, and expressed in µm².