Las af image analysis software

From 8 × 10⁵ to 1.6 × 10⁶ hBM-MNCs were seeded in 2-well culture chamber slides (Thermo Scientific) and cultured in DMEM/10%PhABS for 6 days, as described above. Cultures were then washed twice in D-PBS and MPCs were fixed in paraformaldehyde 4% for 15′. Permeabilization was performed by incubation in Triton X-100 0.5% (Sigma Aldrich) for 15′, after the removal of the fixative by extensive wash in D-PBS. Slides were blocked applying Image-iT™ FX signal enhancer (Thermo Scientific) and incubated with anti- human nestin monoclonal antibody (Abcam, Cambridge, UK) overnight. Antibody excess was removed by washing in Triton X-100 0.5% and slides were then incubated with AlexaFluor® 488-conjugated secondary antibody for 1 h. F-actin staining was performed by AlexaFluor® 555-conjugated phalloidin (Thermo Scientific) for 20′. The slides were finally mounted with ProLong® anti-fade reagent with DAPI and pictures were taken using an inverted fluorescence DM IRB microscope (Leica, Wetzlar, Germany) equipped with LAS AF image analysis software (Leica).

Anesthetized mice were transcardially perfused using PBS followed by 4% paraformaldehyde (PFA). Brains were extracted and post-fixed in 4% PFA overnight. Free-floating 50 µm vibratome (Leica, Wetzlar, Germany) sections were incubated overnight with anti-IBA1 (1:1000, 019-19741, Wako, Japan). Alexa Flour 488-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) were used at 1:200 for 2 h at room temperature. Nuclei were counterstained using 4′6-diamidino-2-phenylindole (DAPI, Roche, Basel, Switzerland) and sections were mounted on glass cover slips. Imaging was performed using the Leica TCS SP8 confocal laser scanning microscope (Leica, Wetzlar, Germany) and LAS AF image analysis software (Leica, Wetzlar, Germany). For DAB (3,3′-diaminobenzidine) staining, peroxidase-conjugated secondary antibodies (Dianova, Hamburg, Germany) were used. Images were captured using A Zeiss AxioImager I (Zeiss, Göttingen, Germany) and the Stereoinvestigator Software 8.0 (MicroBrightField, Magdeburg, Germany).

Free-floating 50-μm vibratome sections from adult brain tissue were stained overnight with anti-IBA1 (1:500) at 4 °C, followed by Alexa Fluor 568-conjugated secondary antibody at a dilution of 1:500 for 2 h at 20–25 °C. Nuclei were counterstained with DAPI. Imaging was performed on a Leica TCS SP8 confocal laser scanning microscope with a ×20 oil immersion objective and the LAS AF image analysis software. Z-stacks with 1.1-μm steps in the z direction, 1024 × 1024 pixel resolution, were analysed using Imaris software (Bitplane). Microglia from cortices (layers 2–5) corresponding to bregma levels −2 to −4 were used for the analysis.