Bz 9000 inverted fluorescence microscope

GFP⁺ microglia were imaged using a 20X / 0.75 NA objective lens on the Keyence BZ − 9000 inverted fluorescence microscope and quantified using the BZ-II Analyzer. Three brain sections per mouse were analyzed. Confocal images of immunohistological preparations were acquired with the SP8 STED-WS (Leica Microsystems) using a HCX PL HCL PL APO C 20X/0.75 NA glycerine objective lens and the LAS X software. DAPI and Alexa Fluors 488 and 647 were excited by the UV Diode Laser 405 nm, Argon Laser 488 nm and WL 647 nm, respectively, and detected in sequential and simultaneous acquisition settings with the HyD detectors in the gating mode. The pinhole was set to one airy unit. Image stacks were sampled with a pixel size of 142 nm and in 1 μm z-steps.

The procedures of immunocytochemistry were described previously^{19 (link)}. Nucleuses were stained with DAPI (1 μg/ml) or PI (2 μg/ml) containing RNase A (50 μg/ml). Samples were examined with a BZ-9000 inverted fluorescence microscope (Keyence). Anti-GFAP rabbit polyclonal antibodies (G4546, Sigma-Aldrich), anti-MBP rat monoclonal antibody (MAB386, Millipore), anti-Ki-67 rabbit polyclonal antibodies (AB9260, Millipore), anti-Hif1α rabbit polyclonal antibodies (NB100-479, Novus Biologicals), anti-Hif2α rabbit polyclonal antibodies (NB100-122, Novus Biologicals), Anti-CDKN2B (p15/INK4b) rabbit polyclonal antibodies (bs-4269R, Bioss Antibody), anti-RUNX1/AML1 rabbit polyclonal antibodies (ab23980, Abcam).

Cells were incubated and treated in slide culture chambers up to the respected endpoint. After washing with PBS, cells were fixed with 4% PFA. After permeabilization with 0.1% Triton-X-100, unspecific epitopes were blocked using 3% BSA in 1× PBS for 75 min at room temperature and subsequently incubated overnight with the primary antibody in 1% BSA in PBS at 4 °C. After washing, cells were incubated with the secondary antibody coupled to the respective fluorophore for 2 h at room temperature (antibodies in Supplementary Table S4). To visualize cell nuclei, 4′,6-diamidine-2-phenylindole (DAPI), staining was performed for 10 min in PBS, and slides were mounted in Aqua Poly/Mount medium. For microscopy, an inverted fluorescence microscope equipped with AxioVision 4.9 software (Zeiss, Jena, Germany) or an BZ-9000 inverted fluorescence microscope (Keyence, Neu-Isenburg, Germany) with automated image stitching function were used.

After cell fixation, cells were stained with Hoechst 33342 (1:500 Sigma-Aldrich, Louis, US) and primary antibodies against vimentin (1:500 mouse mAB ab92547 Abcam, Cambridge, UK), nestin (1:500 mouse MAB353, Sigma Aldrich, Louis, US), tubulin (1:500 rat MAB1864, Millipore, Germany), actin (1:500 Phalloidin Atto 488), Tuj-1 (mouse MAB1864, Millipore, Germany), GFAP (rabbit G9269, Sigma Aldrich, Louis, US), and Sox2 (1:200 goat AD2018, R&D Systems).
Before staining for BrdU, cells were incubated in 2 N HCl for 30 min for antigen retrieval. For visualization, fluorescein-labeled anti-mouse immunoglobin (goat anti-mouse IgG, Alexa Fluor TM 488, Thermo Fisher Scientific, Waltham, USA), anti-rabbit IgG (goat anti-rabbit IgG, Alexa Fluor TM 568, Thermo Fisher Scientific, Waltham, USA), anti-goat (donkey anti-goat IgG, Alexa Fluor TM 568, Thermo Fisher Scientific, Waltham, USA) were used. Cells were counted randomized and manually using ten pictures per trial taken with a Keyence BZ-9000 inverted fluorescence microscope (Keyence Osaka, Japan). Images were taken with a confocal laser scanning microscope (LSM880, Carl Zeiss, Germany) to investigate stress fibers. Imaging by confocal microscopy was also performed to image samples in three dimensions (Z-stack). For 3D reconstruction, Imaris software (Bitplane, Belfast, UK) was used.