Zen 2012

To analyze cardiac myocyte cross-sections, 20 weeks old WT and syndapin III KO mice were killed by cervical dislocation. Beginning at 1000 µm from the apex, hearts were cut in 8 µm thick slices using a cryostat (CM3050S Leica Biosystems).
Cardiomyocytes lining the left ventricle from at least two sections per animal were stained with WGA and cardiac myocyte cross-sections were determined (Fluar 40x/1.30 Oil M27 objective; Zeiss Apotome2/Axio Observer.Z1; AxioCam MR Rev3 camera). The analysis was done using ImageJ software (ImageJ).
Left ventricular wall thickness was determined using cardiac cryosections from 20 weeks old mice stained with H&E that were imaged by using a EC Plan-Neofluar 5x/0.16 objective (microscope and camera as above). Pictures were taken as tiles with 20% overlap. Based on a stitched image (ZEN 2012 software (Zeiss)), the wall thickness was calculated as half the difference of the outer and inner diameter of the left ventricle using the ZEN 2012 software (Zeiss).

Paraffin-embedded sections (5 μm) from mice aorta were rehydrated in several changes of ethanol and Tissue-Clear^® (Sakura Finetek). Sections were then stained for collagen and elastin as previously described [13 (link)]. Sections were studied under light microscopy ZEN 2012 (Zeiss, Jena, Germany). Quantification of collagen and elastin were performed blinded. A scoring system from 1 to 4 was used with 1 defined as no degradation of elastin, 2 as low degradation, 3 as intermediate degradation, and 4 as high degradation. For collagen quantification, 1 defined as low amount of collagen in all layers of the aorta, 2 as low amount of collagen in aorta media or in aorta adventitia, 3 as high amount of collagen in aorta media or in aorta adventitia, and 4 as high amount of collagen in all layers of the aorta. The suprarenal aortic diameter was measured as the adventitial diameter and the definition of an aneurysm was set as a 1.5-fold enlargement of leading edge to leading edge. Measurements of the aortic diameter was performed using ZEN 2012 (blue edition, Carl Zeiss Microscopy GmbH, 2011 ver 1.1.2.0, Oberkochen, Germany). To measure aortic outer diameter, the largest dilated part of the aorta was taken for this purpose.

Confocal microscopy was performed with an inverted Leica TCS SP5 microscope, equipped with lasers for 405, 488, and 561 nm excitation. Images were acquired with a 63×/1.4 NA HCX PL APO objective and image pixel size of 240 nm. The following settings were used for detection: DAPI: 410–480 nm, AlexaFluor-488: 489–560 nm, AlexaFluor-568: 569–640 nm. Image stacks with a z-distance of 0.5 μm per plane were acquired using a 1-Airy-unit pinhole diameter in sequential imaging mode to avoid bleed through. Images shown in Figure 3 were acquired using a LSM 780 confocal laser-scanning microscope with a 40×/1.3 Plan-Apochromat objective controlled by Zen 2012 (Carl Zeiss Microscopy GmbH) in spectral imaging mode. Spectral image stacks were acquired in λ mode using 4 laser lines (405, 488, 561, and 633 nm) and the QUASAR detector (detection range 411–695 nm) with a z-distance of 0.5 μm per plane and the pinhole set to 1-Airy-unit. λ stacks were subsequently linear unmixed with Zen 2012 (Carl Zeiss Microscopy GmbH). Maximum intensity projections were calculated for display purposes and brightness and contrast were adjusted identically for all images of the same dataset using ImageJ/Fiji.

Fluorescence microscopy images were captured in vivo. For MpkC::GFP detection during germination, conidia were germinated for 2, 4, or 7 h at 37°C and observed using confocal microscopy. For stress treatments, 6 h germinated conidia were treated or not with 10 mM H₂O₂ for 10 min and observed within the next 10 min, or were germinated for 6 h in the presence of 1.2 M sorbitol. Image processing and fluorescence quantification were made using Image J and ZEN 2012 (Carl Zeiss, Jena, Germany). To observe conidiophores, the growing edge of a MpkC::GFP colony grown for 3 days at 37°C was sectioned, a drop of water was added and the section was carefully covered with a coverslip. Different fields in which conidiophore structure was better preserved were chosen for observation using confocal microscopy. To observe growing hyphae, 14 h grown mycelia was treated or not with 10 mM H₂O₂ for 20 min or grown for 14 h in MM containing 1.2 M sorbitol and then observed using confocal microscopy. All images were acquired using a Zeiss LSM800 inverted laser scanning confocal microscope using a Plan-Apochromat 63×/1.4 oil immersion objective and 488 and 561 nm laser lines. Maximum intensity projections were obtained from Z-stack images collected every 15 μm through entire cell volume. Images were processed using software ZEN 2012 (Carl Zeiss, Jena, Germany).

MDCK cells stably expressing H2B-mRed were set up for 3D culture in 35 mm glass-bottom μ-Dish (81158, IBIDI). Live imaging was performed with a custom-built spinning disk confocal system based on Zeiss spinning disc confocal microscopy with a spinning disc unit (Yokogawa CSU-X1), Hamamatsu electron-multiplying charge-coupled device camera and humidified, environmental control incubator (37 °C, 5% CO₂). Images were acquired starting ~36 h after virus infection. Image stacks of complete cysts were captured with a EC Plan NeoFluar × 40/0.75 DIC (air) objective every 30 min for a duration of 48 h using Zen 2012 (Carl Zeiss Oy, Vantaa, Finland; Blue edition) software and were analyzed with Zen 2012 (Blue edition).

For lesion quantification, myotubes fulfilling appropriate criteria (thicker than 10 μm, containing laterally-aligned sarcomeres) were randomly selected. In all cases, areas containing lesions were photographed avoiding areas in close proximity (<10 μm) of myotube branching. Images were recorded using a Zeiss Imager M1 fluorescence microscope and Zen pro 2012 (blue edition) (Carl Zeiss, Jena, Germany), using the auto-exposure settings, and processed for quantification as described in the next section.
Confocal images were recorded using a Zeiss LSM 710 and Zen 2012 (black edition) (Carl Zeiss). Live imaging was performed using a Zeiss Cell Observer SD spinning disk microscope and Zen 2012 (blue edition) (Carl Zeiss). For the study of lesion behaviour during contraction, videos of cells transfected with FLNc-EGFP were recorded while the cells were induced to contract tetanically by EPS (as described above) for a few seconds, starting from a relaxed state, thus recording both the relaxed and contracted states. Images of the relaxed and contracted states were exported from the videos and analysed as described in the next section.

Caco2 cells were cultured in 8-well ibidi µ-slides (ibidi GmbH, Martinsried, Germany) and treated with 200 µg/mL of EVs for 2 h at 37 °C, 5% CO₂. After washing with PBS, cells were fixed with 4% PFA and incubated with a buffer solution containing 0.05% bovine serum albumin (BSA), 0.1% Triton X-100, and 0.025% Tween 20 in PBS. Cells were incubated consecutively with mouse monoclonal antibody NheA IgG (1A8) and NheC IgM (3D6) or NheB IgG1κ (1E11) and NheC IgM (3D6) for 1 h at a final concentration of 4 µg per well and were detected with the secondary goat anti-mouse IgG AlexaFluor®488 and goat anti-Mouse AF568 IgM (2 µg per well, respectively) (Molecular Probes, Life Technologies, USA). Nuclei were stained with 1.5 μM 4',6-diamidino-2-phenylindole (DAPI, Sigma Aldrich). Fluorescence 3D-SIM (structured illumination microscopy) images were acquired with the Zeiss LSM710 Elyra PS.1 microscope system equipped with an Andor iXon 897 (EMCCD) camera. Image processing was performed using the Zeiss ZEN 2012 software (Carl Zeiss Microscopy GmbH, Jena, Germany).