Dm6000 epifluorescence microscope

CTR and KO MEF cells were seeded in 8-well ibidi slides (Ibidi GmbH) at a density of 7000 cells per well, stained with MitoTracker Deep Red FM (200 nM for 30 min at 37 °C; Invitrogen) and fixed with 4% paraformaldehyde for 20 min at room temperature. Images were acquired using a Leica DM6000 epi-fluorescence microscope (63 × objective), by using an excitation wavelength of 620 nm and detecting emission using a 670 nm filter (red). Mitochondrial shape was classified as described before^{29 (link)}: (i) category I comprises cells with healthy, elongated and equally distributed mitochondria, which are organized in a tubular network; (ii) category II comprises cells with partially fragmented mitochondria, which are still distributed throughout the cytosol, (iii) category III comprises cells with completely fragmented mitochondria, accumulating around the nucleus. At least 500 cells were counted by an experimenter blinded to the genotype.

Embryos were fixed with 3.7% PFA in PBS overnight at 4 °C, washed in PBS, transferred to 30% sucrose and mounted in OCT for freezing and sectioning. Sections (8–10-μm thick) were postfixed in 3.7% PFA in PBS, permeabilized in methanol for 10 min at −20 ^oC (except for phalloidin staining), then blocked in 5% BSA in 1% BSA 0.1% Triton-X100 (PBT) and stained with antibodies in 1% BSA PBT. The complete list of antibodies and secondary reagents used can be found in the NR reporting summary. The coverslips were mounted using Vectashield containing DAPI (Vector Laboratories). Sections were imaged with Leica DM6000 epifluorescence microscope equipped with HCX PL APO 10.0 × 0.40, HCX PL APO 20 × 0.7 and HCX PL APO 40 × 0.75 objectives or with a Dragonfly 503 spinning disc confocal microscope and/or equipped with a HC PL APO 20 × / 0.75 IMM objective.

Vessels for immunofluorescence were dissected on ice and snap-fixed in ice-cold 4% paraformaldehyde. Paraffin-embedded arteries were sliced at 4 μm, dewaxed and rehydrated. The pFAK Y397 integrin (1:100; AB4803, Abcam) and a nonimmune control IgG antibody were used separately, followed by donkey anti-rabbit antibody with conjugated TRITC (1:200, 1 μg/ml; Jackson ImmunResearch Laboratories, West Grove, Pa., USA). Diamidino-2-phenylindole (DAPI, 1 pg/ml) (Jackson ImmunoResearch Laboratories) was used as a blue fluorescent nuclear stain. A Leica DM6000 epifluorescence microscope was used at ×1,000 magnification in conjunction with Leica FW4000 software for image capture and analysis. Superimposing fluorescence and phase-contrast microscopy images allowed the visualization of the VSMC membrane and the internal elastic lamina which indicates the smooth-muscle/endothelium boundary.

Neurospheres were incubated for 1.5 h at 37°C in 1 mg/ml Texas Red labeled dextran (10 kD Invitrogen), and extensively washed and plated on poly-L-ornithine and fibronectin-coated glass coverslips for 4 h before being fixed in 4% paraformaldehyde. Transfected astrocytes were plated on poly-L-ornithine coated coverslips and scratched 4 d later, followed by the addition of 1 mg/ml fluorescent dextran. Cells were fixed in PFA after 1.5 h and analyzed on the Leica DM6000 epifluorescence microscope described above. The relative amount of dextran uptake was determined using ImageJ to quantify the number of fluorescence maxima per cell after background subtraction.

MYB gene rearrangements were detected on 4-μm-thick paraffin-embedded sections from selected blocks by an ampipine MYB (6q23) gene fracture probe reagent according to the manufacturer’s instructions. A dual-color break-apart interphase FISH assay was performed using centromeric (BAC clone RP11-349 J5; red) and telomeric (BAC clone RP11-641019; green) probes. Probe labeling, hybridization, post-hybridization washing, and fluorescence detection were performed according to procedures established at the Molecular Cytogenetics Core Facility. Slides were scanned using a Leica Dm6000 epifluorescence microscope equipped with cytology imaging software.
If red and green signals were separated by more than two hybridization signals (split-apart signals), or if there were extra single red or green signals in more than 30% of the nuclei in a case, we calculated them as positive. At least 100 nuclei per case were evaluated.

Pups were deeply anesthetized with ketamine (200 mg/kg) and xylazine (20 mg/kg) before transcardiac perfusion with 4% PFA in PBS. Brains were post-fixed for maximum 24 hr, cryoprotected in 30% sucrose in PBS, embedded in 7.5% gelatin/10% sucrose in 0.12 M phosphate buffer, and frozen in isopentane at −50°C. Cryosections of 20 μm or microtome sections of 50 μm were blocked with PBS-GT (0.2% gelatin, 0.25% Triton-X100 (sigma) in PBS) and incubated overnight at RT with the following primary antibodies against Plexin-B2, Pax6, Calbindin (CaBP), Ki67, Tag1, H3P, GFP, Myelin (MOG), Vglut1 as listed in the Key Resources Table. Species-specific Alexa-conjugated secondary antibodies (Jackson ImmunoResearch or Invitrogen) were diluted 1:750 and incubated 1–2 hr at RT. Sections were counterstained with DAPI and embedded in Mowiol. Images were acquired with a DM6000 epifluorescence microscope (Leica) and a laser scanning confocal microscope (FV1000, Olympus) using Fluoview FV10-ASW software (Olympus). Images were reconstructed using FIJI (NIH) or Imaris software (bitplane). Adobe Photoshop was used to adjust brightness, contrast, and levels.