Plan apochromat 63x 1.40 oil dic m27 objective

Opsonization and phagocytosis was measured in an assay using FITC-conjugated conidia and isolated human neutrophils. FITC-conjugation was made by mixing FITC powder (F7250, Sigma-Aldrich) and conidia (5 × 10⁻⁸ μg/conidia) for 30 min end-over-end at room temperature followed by removal of unbound FITC by extensive washing. FITC-conjugated A. fumigatus conidia (1 × 10⁷/ml) were opsonized for 30 min at 37°C in 10% C1q-deficient serum including 10 μg/ml of either the MBL inhibitor (3F8) or mock-inhibitor (1C10). Opsonized conidia were washed and combined with human neutrophils isolated with Polymorphprep (Axis-Shield, Oslo, Norway) according to the manufacturer’s instructions. Neutrophils and conidia co-incubated for 30 min at 37°C in a cell ratio of 1:5. After washing the cells and before flow cytometric analysis, 50 μl tryphan blue was added to quench fluorescence from non-ingested conidia. FITC-positive neutrophils were identified by gating. Barbital buffer was used as dilution/washing buffer throughout the experiment.
We also performed fluorescence and differential interference contrast (DIC) imaging of neutrophils phagocytizing FITC-conjugated A. fumigatus conidia (using the same protocol) to get a visual impression of the process. We used Zeiss LSM 700 Axio Imager 2 with a Plan-Apochromat 63x/1.40 Oil DIC M27 objective and Carl Zeiss ZEN Blue edition software.

The M phase synchronization was performed as previously described^{59 (link)}. In brief, HCT116 cells were treated with 3 mM thymidine for 24 hr, released in fresh medium for 9 hr and then incubated with 0.3 μM nocodazole for 4 hr. Mitotic cells were washed and collected by mitotic shake-off, re-suspended in complete medium and seeded on sterile glass slides. After incubation for 40 min, cells were fixed with −20 °C-stored methanol/acetone (1:1) and subsequently probed with primary and secondary antibodies. Images were acquired with a Zeiss LSM700 confocal microscope using Plan-Apochromat 63x/1.40 Oil DIC M27 objective. 0.5-μm optical sections in the z-axis were collected. The percentage of colocalization was analyzed using Zeiss Zen 2009 light edition software.

To study OGG1 retention under different experimental conditions, a pre-extraction treatment was performed to wash off soluble proteins loosely bound to the chromatin. U2OS cells stably expressing GFP-tagged OGG1 were treated with 0.1% Triton X-100 in PBS (Sigma) for 1 min prior to fixation with 4% paraformaldehyde (Santa Cruz). DAPI was used to stain the DNA for 10 min. Cells were then examined under a Zeiss LSM 780 confocal microscope equipped with a UV-transmitting Plan-Apochromat 63x/1.40 Oil DIC M27 objective. A 488 nm Ar laser was used to excite GFP. OGG1-GFP nuclear fluorescence signal intensities were recorded from three independent experiments. Images were processed in ImageJ and Cell Profiler. Data from at least 1300 cells for each treatment condition were assessed. The mean fluorescence intensity and the standard deviation were calculated and displayed using GraphPad Prism software.

Harvested pellets were snap frozen in Tissue-Plus O.C.T. Compound (Fisher HealthCare) and stored at −80°C until further processing. Samples were sectioned at 5 µm thickness and slides were stored at −80°C until staining. Probe sets for RNA FISH were conjugated with Quasar 670 dye and were synthesized by LGC Biosearch Technologies to detect signal from a congregation of multiple probes binding to target DNA. GAPDH probe set was pre-designed by the manufacturer. Probe sets are listed in Figure 2—source data 2. Staining was carried out according to the manufacturer’s protocol for frozen tissues. Slides were mounted with Prolong Gold anti-fade mountant with DAPI (ThermoFisher) and imaged with the Virtual Slide Microscope VS120 (Olympus) at lower magnification. Confocal microscopy (Zeiss LSM 880) was used to capture images at higher magnification with the Plan-Apochromat 63x/1.40 Oil DIC M27 objective. Fluorescence signal from target RNA FISH probes was captured using a 633 nm excitation wavelength coupled with the Airyscan detector (Zeiss) to achieve the best resolution with improved signal-to-noise ratio (Weisshart, 2014 ). Hoechst signal was captured on the PMT detector utilizing a 405 nm excitation wavelength.

Images were acquired using ZEN black 2009 software operating a LSM 710 confocal laser scanning system (Carl Zeiss MicroImaging Inc., Dunedin, FL, USA) with a Plan-Apochromat 63x/1.40 Oil DIC M27 objective. The cell nuclei were stained with DAPI and measured in the 405 nm channel (MBS -405/760+). GFP-GABARAP was detected in the 488 nm channel (MBS 488), Transferrin (Tf)-Alexa 555 conjugate (T35352, Thermo Fisher Scientific) in the 543 nm channel (MBS 458/543) and EGF-Alexa 647 conjugate in the 633 nm channel (MBS 488/543/633), respectively. HEK293 GFP-GABARAP KI cells (2 × 10⁵) were seeded on fibronectin (F1141, Sigma–Aldrich) coated glass bottom µ-dishes (81158, ibidi), incubated overnight at 37 °C and 5% CO₂ in growth medium. Directly before measuring, medium was replaced by cold medium containing 40 ng/mL EGF-Alexa 647. Data were post-processed using ImageJ [68 (link)] (version: 2.0.0-rc-43/1.50e).