Fluoview 500

The conformation state of β₂-integrins was determined using the activation epitope specific mouse monoclonal mAb24 antibody labelled with Alexa488 (BioLegend, San Diego, California, USA). 5x10⁵ cells were resuspended in 400 μl of RPMI-1640 medium and labelled with mAb24-Alexa488 antibody for 20 minutes on ice under sterile conditions. Without washing, the tubes were moved to a 37°C CO₂ incubator or left on ice (0-minute controls). To induce activation, 100 ng/ml LPS was added to the cells at the beginning of the 37°C incubation. After the incubation time cells were washed with ice cold PBS supplemented with 0.4% sodium azide and 1% FCS and put on ice immediately, to stop the receptor internalization. Samples were analyzed on Cytoflex flow cytometer at 0 minutes and 30 minutes. Confocal microscopy images were prepared at 0, 5 and 30 minutes by Olympus IX81 confocal microscope (60x objective) and FluoView500 software.

Cell cultures were fixed in 4% paraformaldehyde and incubated overnight at 4°C with specific antibodies at indicated dilutions: rabbit anti‐frataxin (1:50) from Santa Cruz Biotechnology (Dallas, USA) and mouse anti‐OxPhos (1:50) from Novex (Invitrogen, Madrid, Spain)). Anti‐rabbit or antimouse secondary antibodies conjugated with Alexa Fluor 555 or Alexa Fluor 488 (Invitrogen, Madrid, Spain), respectively, were used at 1:300 dilutions and incubated for 2 hrs at room temperature, protected from light. For cell nucleus staining, the fixed cells were incubated for 5 min. with DAPI. Micrographs were taken using an Olympus FluoView 500 (60×).

At the 8th week, immunohistochemistry was used to detect the osteogenic factor osteocalcin (OCN, abcam-93876) and angiogenic factor VEGF (abcam-72807)), while the immunofluorescence was used to detect the molecular of the signal pathways including p-AKT (abcam-222489), HIF-1α (abcam-8366), GSK-3β (abcam-226169), β-catenin (abcam-184919), RANKL (abcam-45039), and p-NFATc1 (MA3-024, Affinity BioReagents). Briefly, the sections were fixed and treated with 50 μg ml−1 4′,6-diamidino-2-phenylindole (DAPI) for nuclear staining. The primary antibodies were diluted to the optimal concentration. The sections were then stained with anti-antibodies and visualized with a secondary antibody conjugated with Cy5 or without Cy5 for immunohistochemistry. Immunohistochemistry images were obtained using a light microscope (BX41, Olympus, Japan). Meanwhile, fluorescence images were acquired using a fluorescence microscope (FluoView 500; Olympus, Tokyo, Japan).
For the semiquantitative analysis of the protein expression, we evaluated the percentage of positive cells (4 scores) and color intensity (3 scores) in immunohistochemistry pictures [23 (link)]. Immunofluorescence pictures were analyzed using Image-Pro Plus software to measure protein expression area and cell elongation ratio [24 (link)].

Histological sections were blocked for 1 h at RT in a PBS containing 0.1% Triton-X (Sigma Aldrich) solution added with 5% normal donkey serum (NDS, Vector Labs) and incubation with specific antibodies against microglial/monocyte markers (Iba1; 1:500, Wako, CD68, F4/80 and CD11c; 1:100, AbD Serotec), neuroblasts (DCX; 1:1000, Millipore) and astroglial lineage cells (GFAP; 1:250, Sigma Aldrich) was performed overnight at 4°C. Proliferative cells were revealed by using an anti-BrdU antibody (1:100, AbD Serotec). To allow labeling of nuclear DNA, before blockage, sections were treated for 1 h with HCl 1M (RT) under agitation (Tang et al., 2007 (link)). Staining was revealed by 2-hour incubation period (RT) with appropriated secondary antibodies conjugated to Cy3 or Cy5 fluorophores (1:250, Jackson ImmunoResearch). DAPI (4′,6-Diamidino-2-phenylindole, 1:1000, Sigma Aldrich) was used for nuclear counterstaining and slides were mounted with ProLong Antifade (Life Technologies). Immunolabeled brain sections were analyzed and imaged using a confocal microscope (Olympus FluoView 500) with 40x (NA 1.30) and 60x oil-immersion (NA 1.25) objective lens (Olympus). Acquired images were adjusted for brightness and contrast using FIJI/ImageJ software.

Shoot apices of the different segregant lines of DR5::VENUS were collected and the meristems were visualized under a confocal microscope. Images were acquired using the Olympus IX81 inverted laser scanning confocal microscope (Fluoview500) equipped with a 488-nm argon-ion laser. VENUS protein was excited by 488-nm light and the emission was collected using a BA 505–525 filter. A BA 660 IF emission filter was used to observe chlorophyll autofluorescence. Confocal optical sections were obtained in increments of 0.5 to l μm. The images were color-coded yellow for VENUS and magenta for chlorophyll autofluorescence.