To examine liposomes infiltration to the mucus, mucus and Cy5-labeled liposomes were imaged under confocal microscopy, with and without NAC treatment. Mucin from the porcine stomach was dissolved to a concentration of 43.75 mg/mL in PBS and gently shaken overnight in RT to form a disulfide bond and form mucus. The mixture was let to achieve relaxation for 24 h. 10 mg/mL NAC was dissolved in PBS for NAC treatment. 8 μL of synthetic mucus was dripped on microscope slide. 2 μL of 10 mg/mL NAC treatment was dripped on top. After 1 h of incubation, 2 μL liposome were placed on top of the described set. A coverslip was placed on top with spacers that ensured that the coverslip was evenly spaced. PBS solution used as control. Images were taken using Zeiss LSM 700 Confocal laser scanning microscopy, and were processed using Zen 3.4 Blue edition.
Zen 3.4 blue edition
Manufactured by Zeiss
ZEN 3.4 blue edition is a comprehensive microscopy software platform developed by Zeiss. It provides an integrated solution for image acquisition, processing, and analysis across various microscopy techniques. The software offers a user-friendly interface and a range of advanced tools to support scientific research and clinical applications.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Axio zoom v16, by Zeiss (2 mentions)
Lsm700 confocal laser scanning microscopy, by Zeiss (1 mentions)
Axio observer z1 inverted, by Zeiss (1 mentions)
Eclipse ni microscope, by Zeiss (1 mentions)
Apotome 2 microscope, by Zeiss (1 mentions)
5 protocols using zen 3.4 blue edition
1
Mucus-Liposome Infiltration Imaging
2
Quantifying dystrophin-positive revertant fibers
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Q muscles were embedded in optimum cutting temperature medium (OCT; VWR International, Dublin, Ireland) and frozen in liquid nitrogen chilled isopentane (VWR International). A total of 10 µm transversal cryosections were fixed with 4% paraformaldehyde and blocked with 10% goat serum. Antibodies directed against dystrophin and laminin were used (Table S1 ). Secondary antibodies were AF488 and AF647-conjugated goat anti-rabbit or anti-rat (Sigma-Aldrich, St-Louis, MO, USA), respectively. Finally, nuclei were stained with DAPI (Thermo Fisher Scientific). Whole slides were scanned with a fluorescence microscope (Axio Scan.Z1, Zeiss, Oberkochen, Germany). Dystrophin-positive fibres were counted and expressed as the percentage of the total number of fibres per muscle (ZEN 3.4 blue edition, Zeiss). A dystrophin-positive revertant fibre (RF) was scored when more than half of its membrane circumference expressed a green positive signal [27 (link)]. The number of clusters (with at least two adjacent RFs) and the maximal number of RFs in a cluster were counted on whole sections as well [27 (link)]. Centrally nucleated fibres (CNF) were also calculated using a personalized version of MuscleJ [28 (link)] based on the automatic detection of laminin and DAPI immunofluorescence.
3
Fluorescence Microscopy Image Optimization
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Fluorescent microscopy images were obtained at the same laser intensities and exposure times using either a Nikon Eclipse Ni microscope (×20 objective) or a Zeiss Axio Observer Z1 inverted microscope (×10 and ×20 objective). Images obtained with the latter were further processed on the Zeiss Zen 3.4 (blue edition) software to improve their clarity via the “Deblurring” function.
4
Fluorescent Imaging of Planula Larvae
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GFP and Rhodamine fluorescent imaging as well as bright field imaging, including in situ hybridization imaging, were taken with an Axio Zoom V16 (ZEISS), and image processing was done with ZEN 3.4 blue edition (ZEISS) and Fiji/ImageJ software45 (link). Intensity of mean values of green fluorescence was measured using the average brightness (pixel value) of the pixels. To prevent planula movement, we added 0.5 M sodium azide (final concentration was less than 0.25 M).
5
Quantitative Analysis of NET Release
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To quantify NET area release, three random images from three independent assays were analyzed from each sample with an ApoTome II microscope using Carl Zeiss ZEN 3.4 (blue edition) software. A machine learning tool was designed for the detection and quantification of NETs, as previously described, with slight modifications [23 (link)]. Briefly, to perform a homogeneous analysis, we chose only fields with 80 nuclei-stained samples with IP. A deep learning module Intellesis Trainable Segmentation from Carl Zeiss ZEN 3.4 (blue edition) for the NET area quantification was used, excluding nuclei and background. The result was considered as the total area occupied by the NET release; data were expressed as mean ± SE.
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