Ix81 spinning disk confocal microscope

Tumors were harvested and fixed in 10% neutral buffered formalin for 24 hours at room temperature and then replaced with 75% alcohol. Tumors were processed in a conventional tissue processor and embedded into paraffin wax using graded alcohol, xylene and paraffin wax. Serial sections of 5 μm were cut from the paraffin embedded tumor blocks for immunohistochemical analyses.
For hypoxia and proliferation analysis, mice were injected i.p. with 2-nitroimidazol hypoxia marker EF5 (30 mg/kg) and active DNA synthesis marker EdU (10 mM), respectively, 2.5 hours prior to sacrifice. For EdU staining the Click-iT cocktail was applied for 30 min at room temperature. Primary antibodies used were as follows: PECAM-1 (Santa Cruz sc-1506), Cleaved Caspase-3 (Cell Signalling), 53BP1^Ser1778 (Cell Signalling), Vimentin (Fitzgerald 20R-VP004), E-cadherin (BD Biosciences).
Secondary antibodies were: Alexa Fluor 488 Donkey Anti-Rabbit (Life Technologies), Biotin-Goat Anti-Guinea Pig IgG (Jackson ImmunoResearch), Biotin-Goat Anti-Mouse IgG (Invitrogen)
Images were acquired using an Olympus IX81 Spinning Disk Confocal Microscope using an PlanApo 60x/1.42 oil objective lens. A minimum of 50 cells were imaged for 53BP1^Ser1778 analysis. Images were processed and analyzed using ImagePro Plus.

A549 cells and Sertoli cells on coverslips were fixed for 15 min at room temperature with freshly prepared 4% paraformaldehyde (Electron Microscope Sciences) in PBS. Samples were then washed three times with PBS, permeabilized with 0.5% Triton X 100 in PBS for 5 minutes at room temperature, washed three times with PBS and incubated in blocking buffer (5% bovine serum albumin [BSA; Sigma Aldrich] in PBS) at room temperature for 1 h. Incubations with primary antibodies in blocking buffer were carried out at room temperature for 1 h, followed by three washes in PBS. Samples were then incubated with corresponding secondary antibodies in blocking buffer for 1 h at room temperature, followed by three washes in PBS. The secondary antibodies (Invitrogen) were used at 1:1000 dilutions in blocking buffer. Prior to mounting, samples were incubated with DAPI (4′,6-diamidino-2-phenylindole; Sigma Aldrich) (1 μg/ml) for 5 min at room temperature before washing. Coverslips were mounted on microscope slides using Prolong Gold anti-fade mounting reagent (Life Technologies). Images were acquired using an Olympus IX-81 spinning-disk confocal microscope equipped with a 40x/1.42-numerical-aperture oil PlanApo N objective. Images were analyzed using Volocity 6.2.1 software (PerkinElmer).

HEp-2 cells were seeded to No. 1 glass coverslips (1014355117NR1, Thermo Fisher Scientific) for high-resolution confocal microscopy, or black, flat, microclear-bottom 96-well plates (655096, Greiner Bio-One) for high-throughput quantification of RSV F and N immunofluorescence. Cells were infected with RSV A2 (MOI 0.5). At 48 h p.i., cells were fixed in 4% paraformaldehyde for 30 min and permeabilized with 0.3% Triton X-100 for 10 min at room temperature. F and N proteins were probed using mouse anti-RSV F (RSV3216) and mouse anti-RSV N (RSV3132) mAbs (Bio-Rad Antibodies) diluted 1:500 in PBS + 0.1% Tween-20 (PBST), or mouse anti-RSV F mAb Alexa Fluor 488 conjugate (Millipore Sigma, 133/1H) diluted 1:200 in PBST over a 1-h incubation at room temperature. This was followed by detection with secondary anti-mouse Fab fragments conjugated to Alexa Fluor 488 (4408) or 647 (4410) (Cell Signaling Technology) diluted 1:1000 in PBST over a 1-h incubation at room temperature. Fluorescent images were acquired using either the Cellomics ArrayScan VTI HCS Reader at ×10 magnification (for quantification) or Olympus IX81 Spinning Disk Confocal microscope at ×60 magnification with oil immersion (for high-resolution images). F and N immunofluorescence was quantified in CellProfiler and normalized to total nuclear count.