Orca flash4.0 lt

Age-synchronized animals at day 1 of adulthood (4-day old animals) were mounted on 3% agarose pads and immobilized using 0,5 mM levamisole in M9. Animals were imaged with a Zeiss Axio Imager.Z2 upright epifluorescence microscope connected to an Orca Flash 4.0 LT, 4 megapixel monochrome sCMOS camera (Hamamatsu) and Zen 2 pro software (Zeiss). Images were acquired with 40x 0.75 NA EC Plan Neofluar objective. Micrographs were processed with Photoshop CS6 software (Adobe Systems).

Capsule was induced using defined low-iron capsule-inducing medium (CIM) prepared as previously described (75 (link)). Briefly, cells were grown overnight in YPD and washed in low-iron water, and 10⁶ cells per ml were inoculated in CIM. Drug-treated cells were grown in the presence of 3 μg/ml antimycin A or 5 μM myxothiazol as indicated. Cells were imaged after 48 h of growth at 30°C with India ink staining using a Zeiss Plan-Apochromat 100×/1.46 oil lens on a Zeiss Axioplan 2 microscope. Images were obtained using an ORCA-Flash4.0 LT digital CMOS (complementary metal oxide semiconductor) camera (Hamamatsu, Hamamatsu City, Japan) and Zen software (Zeiss, Oberkochen, Germany). The capsule size was measured for 50 cells from each strain using ImageJ (76 (link)), and the difference in capsule size between strains was evaluated using a Kruskal-Wallis analysis of variance (ANOVA) in GraphPad Prism 6.0 (GraphPad Software, San Diego, CA).

Fetus sections were rehydrated in PBS before antigen retrieval treatment in a pH6 citrate buffer solution at 95°C for 15 min followed by 20 min of cooling. They were blocked with 0.5% Triton complemented with 5% horse serum for 1-3h at RT. Sections were incubated with primary antibodies at +4°C overnight, then with secondary antibodies for 45min-1h at RT; antibodies were diluted in the blocking solution. For s1s4s2s5KO fetuses, Pax7 immunostaining required different steps: sections were permeabilized with cold -20°C acetone for 10 min, air-dried 10 min, then blocked and incubated with antibodies as previously explained. Six1 immunostaining required an amplification step using a biotinylated secondary antibody. Sections were incubated with horseradish peroxidase (HRP)-conjugated streptavidin for 30 min and treated with Alexa Fluor 488 tyramide for 10 min (SuperBoost tyramide signal amplification kit; Thermo Fisher Scientific). Immunostained sections were mounted in Mowiol mounting medium before imaging. Images were taken on either an upright fluorescent microscope (Olympus BX63), equipped with an ORCA-Flash4.0 LT Hamamatsu camera, using Metamorph 7 software or with an inverted fluorescent confocal microscope (IXplore Spinning IX83) equipped with a Hamamatsu sCMOS Orca flash 4.0 V3 camera and using CellSens Dimension software. See Table 1 for primary antibodies references.

To visualize the free DNA present in neutrophil-planktonic co-cultures, neutrophils were pre-stained with Calcein AM (Thermo Fisher Scientific, Waltham, MA, USA) at 0.5 μg/mL in DPBS at room temperature for 10 min in the dark and added at 2 × 10⁶ cells/mL to 3 × 10⁷ planktonic cells in µ-Slide 8-well plates (ibidi, Martinsried, Germany) [19 (link)]. Fifteen minutes prior to imaging, propidium iodide was added at 3 µM. Fluorescent images were obtained (excitation 480 nm/emission 525 nm and excitation 565 nm/emission 620 nm) with a 20× objective on an inverted microscope (Nikon Eclipse TE300) equipped with a charge-coupled device camera (CoolSNAP ES2) and MetaVue imaging software v6.2. Images were processed using ImageJ. For citrullinated histone detection, we used anti-histone H4 (citrulline 3) antibody (Millipore, Billerica, MA, USA) as described previously [19 (link)]. Images were acquired using brightfield and fluorescent (excitation 565/emission 620) detectors with a 40× objective on an inverted microscope (TI2-E, Nikon, Tokyo, Japan) equipped with a sCMOS camera (Orca-Flash 4.0 LT+, Hamamatsu, Hamamatsu City, Japan) and NIC Element imaging software.