Axiocam mrm rev 3

For localization of AnCtrA and AnCtrC, conidiospores of GFP-tagged strains were cultured in Ibidi μ-dishes, 35 μm high (Ibidi GmbH, Germany; 2 μl of medium per well) for 16 h at 30°C in liquid Aspergillus Minimal WATCH medium (Penalva, 2005 (link)) adequately supplemented and containing 0.1% D-glucose, 71 μM sodium nitrate, 25 μM sodium phosphate monobasic. For low copper availability conditions, 100 μM bathocuproine disulfonic acid (BCS) Cu chelator were added to the WATCH media.
Fluorescence microscopy was performed using a Zeiss Axio Observer Z1 inverted microscope (63 Plan Apochromat 1.4 oil immersion Lens) and Axiocam MRm Rev.3 camera. For observation of GFP, a filter set 38 HE (Ex BP 470/40; FT 495; Em BP 525/50) was used. The images shown are representative of at least five experiments repeats. Levels of fluorescence were analyzed using open source ImageJ software¹⁰ (U. S. National Institutes of Health, Bethesda, MD, United States).

Imaging was performed using standard fluorescence microscopy with a Zeiss Axio Observer.Z1 inverted microscope (Carl Zeiss, Oberkochen, Germany), equipped with an AxioCam MRm Rev.3 camera (Zeiss) and a Plan-Apochromat 100x/1.40 objective (Zeiss).

For histological evaluation, harvested tumor tissue was fixed in 2% formalin (neutral buffered) and embedded in paraffin. Thin tissue slices (~5 µm) were cut using cryotome, and the sections were mounted for antigen retrieval. Standard immunohistochemistry steps of deparaffinizing and rehydrating with various solvents was followed, and the immunostaining (Leica Bond automated stainer) was carried out. Primary and secondary antibodies were used for CD34 (Abcam ab8158 / 1:100 and HRP) and phosphohistone gammaH2AX: ser139 staining (Cell signaling technologies; #20E3 / 1:400). Sections were stained and counterstained with Mayers hematoxylin. Following blocking and DAB steps, images were visualized using a Zeiss Axio Imager M2 microscope with a high-resolution Axiocam Mrm Rev.3 camera at 20x and 100x magnifications.

HCoEpC were grown on cover glasses, treated with Benzo[a]pyrene (5 µM) and/or Hydroxytyrosol (1 µM) for 6 days, and mitochondrial labelling was performed by adding MitoTracker^TM Green-FM (Invitrogen, Cat# M7514, MA, USA) fluorescent dye (200 nM) to cell culture medium for the last 30 min at 37 °C. Then, cells were washed in PBS and cover glasses mounted with PBS:Glycerol (1:1) on microscope slides. Cells were analyzed with Apotome Axio Observer Z1 inverted microscope (Zeiss, Munich, Germany) equipped with an AxioCam MRM Rev.3 (Germany) at 40× magnification.

Jurkat T cells and primary T cells were loaded with Fluo-4/AM (1 µM) in serum-free RPMI-1640 media at room temperature for 30 min. Afterward, cells were pelleted by centrifugation, followed by one wash using Ringer’s solution containing 0 mM Ca²⁺. Then cells were settled on the motor-linked substrates for 8 min before measurement. Afterward, the cells were washed once with Ringer’s solution containing 0 mM Ca²⁺. Then the same volume of Ringer’s solution containing 2 mM Ca²⁺ was added prior to the start of calcium imaging. Fifteen minutes later, UV illumination was conducted for 1 min, followed by another 15 min of imaging. To detect a general capability of the cells to induce Ca²⁺ influx, 1 µM thapsigargin is added 10 min after UV illumination.
For calcium imaging, we employed a Zeiss Cell Observer HS system with a ×20 alpha objective and an AxioCam MRm Rev. 3. During the experiment, the fluorescence of Fluo-4 was acquired with a 38HE filterset every 5 s. Mean fluorescence intensity of Fluo-4 was quantified with ImageJ. The cell spreading areas were determined using the Wand (tracing) Tool in ImageJ and then quantified with ImageJ. The peak of Ca²⁺ influx (ΔPeak) is the maximum relative the fluorescence of Fluo-4 (normalized to baseline before UV illumination) from first UV pulse to 120 s.