Fluoview 3

Immunofluorescence at replication structures and whole nuclei was visualized using an Olympus FV1000 confocal BX61 upright microscope equipped with X 60(1.42 NA) or X 100 (1.4 NA) objective lens, respectively. Images were captured and analyzed by Fluoview 3.1 software (Olympus, Shinjuku, Tokyo, Japan). Measurements of labelled tracks were performed in micrometres, by using the ImageJ software (http://rsbweb.nih.gov/ij/), and converted to kilobases with the reported(30) factor 1μm=2.59Kb.
For co-localisation experiments, serial optical sections of 0.2μm were collected through nuclei using a wide-field fluorescence microscope (DeltaVision DV3; Applied Precision, Issaquah, WA, USA) equipped with a 100X (1.4 NA) objective (Olympus). SoftWorx (Applied Precision) software was used to deconvolute image stacks, which were further analysed with Volocity (Perkin Elmer, Waltham, MA, USA) software. Nuclear foci with size >0.05 and <1.0μm, labelled with CIdU and/or IdU were measured in each cell.

Eyes were enucleated from WT and hph-1 mice at P22 and then fixed in 4% paraformaldehyde at room temperature for 2 h. Eyes were incubated  overnight at 4 °C in a 30% sucrose solution prior to embedding in OCT compound (TissueTek®). Coronal sections of the eyes were cut at a thickness of 10 μm by using a Cryostat (Leica). Sections were subsequently washed with PBS, blocked and permeabilized for 1 h at room temperature and subsequently incubated overnight with Lectin from Bandeiraea simplicifolia TRITC conjugate (Sigma; 1:100) for retinal vasculature and/or antibodies to rabbit Iba-1 (Wako Chemicals USA, Inc.; 1:400), goat IL-1β (R&D systems; 1:300), or rabbit anti-CD36 antibody (Abcam; 1:200). The primary antibodies were labeled for 2 h with Alexa-488 goat anti-rabbit or donkey anti-goat IgG obtained from Molecular Probes (Eugene, OR) and used at dilutions of 1:1000. Samples were visualized using 30× objective with an IX81 confocal microscope (Olympus), and images were obtained with Fluoview 3.1 software (Olympus).

Cells were seeded in 6-well plates in media containing inhibitors. After 24 h 25 μM (final concentration) of CldU (Sigma) was added to cells for 20 min at 37 °C before 250 μM IdU (final concentration, Sigma) was added for a further 20 min at 37 °C. DNA fibre analysis was then carried out as previously described [14] (link). Immunofluorescence was visualised using an Olympus FV1000 confocal BX61 upright microscope equipped with × 60 (1.42 NA) objective lens. Images were captured and analysed by Fluoview 3.1 software (Olympus, Shinjuku, Tokyo, Japan). Measurements of labeled tracks were performed in micrometres, by using ImageJ software (http://rsbweb.nih.gov/ij/), and converted to kilobases with the factor 1 μm = 2.59 kb.

For experiments using a single dye individual neurons were viewed at their soma equator with a Nikon Eclipse TE 200 inverted microscope through a Nikon 100X oil-immersion objective (NA 1.40). Fluorescence measurements of FM1-43 or Fluo-4 were performed with excitation and emission wavelengths at 488 and 535 nm respectively. A cooled CCD camera (IMAGO, Till Vision) acquired image sequences of 640 × 480 pixels. To study fast Fluo-4 fluorescence transients in response to the stimulation trains, images were acquired every 100 ms for 60 s. To follow FM styryl dye changes or the long-lasting Fluo-4 transients, separate images were acquired every 2 s for 15 min. The image sequences were stored digitally by using TILLvisION software.
For simultaneous recordings of exocytosis and Ca²⁺ signals, confocal imaging of FM4-64 and Fluo-4 fluorescence was carried out using an Olympus Fluoview FV1000 upright confocal scanning microscope using 473 nm for Fluo-4 excitation and 560 nm for FM4-64 excitation. For the simultaneous imaging fluorescence was acquired with a 60X water immersion objective (1.1 NA). Fluorescence was detected in parallel by a spectral-based detector capturing 503–543 nm for the Fluo-4 emission and 600–700 nm for the FM4-64 emission. Time-lapse sequences were made by acquiring an image every 2 s for 20 min. Images were stored digitally by using Fluoview 3.1 software (Olympus).

Eyes of P17 C57BL6/J pups exposed to normoxia or OIR were enucleated, fixed in 4% paraformaldehyde for 1 h at room temperature, and saturated overnight at 4°C in a 30% sucrose solution prior to embedding in OCT compound (TissueTek^®). Sagittal cross-sections of 10 μm was sectioned using a Cryostat (Leica) and permeabilized for 1 h at room temperature. Immunostaining against Sema3E (R&D Systems; 1:100), NeuN (EMD Millipore; 1:100), F4/80 (Abcam; 1:100), or IL-17A (Abcam; 1:200) overnight at 4°C, followed by fluorochrome-conjugated secondary antibody (goat anti-mouse IgG Alexa Fluor 488 and goat anti-rabbit IgG Alexa Fluor 594; Invitrogen) for localization studies according to manufacturers’ recommendations. Nuclei were stained with DAPI (Invitrogen; 1:5000). Cross-sections were visualized using 30× objectives with an IX81 confocal microscope (Olympus), and images were obtained with Fluoview 3.1 software (Olympus).

Fluorescent images were obtained with a Hamamatsu (Orca C4742-95) camera coupled to the Olympus image acquisition system Cell M (excitation light 450–490 nm through a dichroic filter; emission light 502–538). Some slices were fixed and covered with Vectashield (Vector Laboratories, Burlingame, CA) for reconstruction. Visualization was made through a confocal fluorescence microscopy (Olympus Fv-1000) and acquired with the OLYMPUS FLUO VIEW 3.1 software.

Cells were washed with and fixed in 2% formaldehyde for 15 min at 37°C. Cells were blocked and permeabilized (1.5% BSA, 5% goat serum, and 0.2% triton X-100 in PBS) for 1 h at 37°C. Antibodies (1/100) were incubated at 4°C overnight and AlexaFluor-conjugated secondary antibodies (1/500 in blocking buffer) incubated for 1 h at room temperature. AF647-conjugated Phalloidin (1/200) was added in PBS containing 1% goat serum and incubated for 20 min at room temperature. Slides were mounted in ProLong antifade Gold containing DAPI (Life Technologies). Frozen tissue section (8 µm) were fixed and stained as described for cells. Slides were imaged using an Olympus IX81 inverted confocal microscope and analyzed using Olympus FluoView 3.0 and quantified using ImageJ.