For the experiment with conidia, germ tubes, and appressoria, we used the wild-type, mutant ∆Cglac13H and the complementary strain C-∆Cglac13H in 100 mL potato dextrose broth (PDB) and cultured by shaking a conical flask for 3 days at 28 °C, at 180 rpm in the dark. We observed the quantity of conidia on the hemocytometer, and then we collected the conidia, diluted them with sterilized water, and adjusted them to 2 × 106 conidium/mL. The conidia suspension of each strain was placed on a glass slide, and we observed the changes in the conidia at 0, 2, 4, 6, 12, 24, and 36 h with a DM2500 Leica microscope (Leica, Wetzlar, Germany). We used cellSens Dimension 1.11 Software to calculate the conidia germination rate and appressoria formation rate. In addition, we observed the germ tubes of wild-type and mutants at 12 h, statistically analyzed the number and proportion of germ tubes in these two strains and imaged the results.
Leica dm2500 microscope
Manufactured by Leica camera
Sourced in Germany, China, United Kingdom, United States, Japan
The Leica DM2500 is a compound microscope designed for laboratory use. It features a sturdy, ergonomic design and offers high-quality optical performance. The microscope is equipped with various objectives, allowing for magnification of specimens. The DM2500 is suitable for a range of applications in fields such as biology, materials science, and clinical research.
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Lab products found in correlation
Las x software, by Leica camera (4 mentions)
Dfc420 camera, by Leica camera (3 mentions)
Leica application suite v3, by Leica camera (3 mentions)
Invia raman microscope system, by Renishaw (3 mentions)
Jem 2100, by JEOL (2 mentions)
Lsm 710 laser confocal microscope, by Zeiss (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dfc425c camera, by Leica camera (2 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (2 mentions)
India ink, by BD (2 mentions)
Axiocam mrc, by Zeiss (2 mentions)
Leica dm 2500 fluorescent microscope, by Leica camera (1 mentions)
Syto 24 green fluorescent nucleic acid stain, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Goat serum, by Vector Laboratories (1 mentions)
Mca519g, by Bio-Rad (1 mentions)
Gill no 2, by Merck Group (1 mentions)
Immpress hrp anti rabbit, by Vector Laboratories (1 mentions)
92 protocols using leica dm2500 microscope
1
Conidial Morphogenesis in Fungal Mutants
2
Measuring Focal Adhesion Morphology
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For analysis of FAs, the morphology of immunostained vinculin was evaluated by measuring the length and the total number of FAs per cell. In particular, the length of individual FAs was measured from images taken at 100× magnification by drawing the longitudinal axis of at least n > 100 vinculin positive signals, collected in 15 different microscope fields (DM2500 Leica microscope, Leica Microsystems, Milan, Italy). The total number of cell focal adhesions was measured on all cells in 10 representative fields collected at 20× magnification (DM2500 Leica microscope). Morphometric analyses were performed in triplicate. All data were analyzed using ImageJ software 1.52r (ImageJ, USA). The frequency distribution of the collected values (length and total number of FAs) was reported as a Gaussian function.
3
Microscopic Examination of Trypanosomatid Cells
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The trypanosomatid cells on Giemsa-stained smears were photographed using DM 2500 Leica microscope and measured with Fiji software [67 (link)]. For fluorescent microscopy, cells from either 3-day-old culture or the intestinal contents of flies infected 3 days before were centrifuged, fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min, rinsed with PBS, attached to poly-L-lysine-coated slides, permeabilized with 1% Triton X-100, and blocked with 1% Bovine Serum Albumin (Sigma-Aldrich). For the visualization of axonemes, primary mouse monoclonal anti-α-tubulin and secondary anti-mouse IgG-TRITC goat antibodies (1:500 and 1:100, respectively; both from Sigma-Aldrich) were used. After washing with PBS and staining with either DAPI (4′,6′-diamidino-2-phenylindole; Sigma-Aldrich) or SYTO24 Green Fluorescent Nucleic Acid stain (Thermo Fisher Scientific), slides were observed under Leica DM2500 fluorescent microscope. The samples from cultures and midguts of infected flies were prepared and processed for scanning and transmission electron microscopy as described previously [8 (link), 68 (link), 69 (link)].
4
Quantifying Aortic Lesions and Components
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Smooth muscle cells were stained with anti-α-smooth muscle actin (SMA; ab5694, Abcam), which detects smooth muscle α-actin 2 (ACTA2), and macrophages were stained using macrophages/monocytes antibody (MOMA-2; MCA519G, Bio-Rad). Slides were fixed in cold acetone at −20°C and air dried, and endogenous peroxidase activity was blocked in 0.3% H2O2/methanol. Nonspecific binding was reduced by incubation in 2.5% goat serum (Vector Laboratories) before primary antibody incubation at 4°C in a humidified chamber overnight. Slides were incubated with ImmPRESS HRP anti-rabbit (Vector Laboratories, MP-7451) or anti-rat IgG peroxidase (Vector Laboratories, MP-7444), and staining was visualized using DAB and counterstained in hematoxylin (Gill No. 2, Sigma-Aldrich). Images were acquired with a DM2500 Leica microscope. Positively stained areas of each aorta were measured with ImageJ by a researcher blinded to genotype. The data are presented as the average of positively stained lesion areas (9 serial sections) normalized by total lesion area. For collagen and lipid core quantification, sections were stained with Masson’s trichrome (Sigma-Aldrich) according to the manufacturer’s instructions.
5
Isolation and Analysis of Adipose SVF
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Visceral white adipose tissue (WAT) was excised from mice and processed for SVF cell isolation as described (16 (link)). Briefly, tissues were cut into small pieces and digested in 1 mg/ml collagenase P (Roche) in HBSS for 40 min at 37°C. The digested tissues were passed through a 100-μm cell strainer to remove debris. After centrifugation, the floating cell layer and supernatant were removed and the cell pellet was washed with HBSS. Primary SVF cells were maintained in DMEM plus 10% FCS. For histology, 3–4 μm of paraffin-embedded sections of WAT were stained with hematoxylin and eosin and analyzed by light microscopy. For quantification of adipocyte size, sections were analyzed by a DM2500 Leica microscope equipped with Leica DFC420C digital camera (Leica microsystem). Adipocyte diameters were measured in 30 adipocytes per section (five sections for each WAT sample), and data analysis was performed using Leica Application Suite (LAS v3.8, Leica microsystems) for digital image processing.
6
Quantifying Pharyngeal Tooth Morphology
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Dorsal and ventral pharyngeal tooth number was quantified on a DM2500 Leica microscope using a TX2 filter as previously described [19 (link)]. For both ventral and dorsal tooth counts, total tooth number equals the sum of the left and right sides (of ventral and dorsal pharyngeal teeth, respectively). Tooth plate area and spacing of the ventral pharyngeal tooth plate were quantified from a gray scale image taken with a DFC340 FX camera on a Leica M165FC as previously described [19 (link)]. Area and spacing of the ventral pharyngeal tooth plates are the averages of the left and right tooth plate. Skeletal traits were binned by total fish length for three stages: early juvenile <27 mm, late juvenile 27–37 mm, and adults >37 mm.
7
Raman Mapping of Microscopic Samples
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Raman spectra were obtained in the backscattering geometry with a Renishaw Invia Reflex Raman spectrometer equipped with a DM 2500 Leica microscope (100× objective, NA = 0.85) in confocal mode, a 600 grooves/mm grating, edge filter, and a 633nm excitation line of a He-Ne laser (power 10mW).
The spectra were acquired over a rectangular zone of 2160 points with a step distance of 1 µm. The exposure time for each point was 20s, leading to a total acquisition time of 12 hours. Data treatment and Principal Component Analysis (PCA) were performed with Renishaw Wire 3.4 software.
The spectra were acquired over a rectangular zone of 2160 points with a step distance of 1 µm. The exposure time for each point was 20s, leading to a total acquisition time of 12 hours. Data treatment and Principal Component Analysis (PCA) were performed with Renishaw Wire 3.4 software.
8
Lipid Droplet Visualization via Nile Red Staining
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Microscopy studies and Nile Red staining were conducted, according to Miranda et al. [27 (link)].
Briefly, lipid staining with Nile Red was achieved by adding 5 µL of stain to a cell suspension and further incubating at 37 °C for 5min. The stained cells were then subjected to fluorescent microscopy analysis to observe the presence of lipid droplets using a Leica DM 2500 microscope with an attached camera Leica DFC 310 FX. Nile Red filter: excitation at 543 nm, emission 555–650 nm.
Briefly, lipid staining with Nile Red was achieved by adding 5 µL of stain to a cell suspension and further incubating at 37 °C for 5min. The stained cells were then subjected to fluorescent microscopy analysis to observe the presence of lipid droplets using a Leica DM 2500 microscope with an attached camera Leica DFC 310 FX. Nile Red filter: excitation at 543 nm, emission 555–650 nm.
9
Histopathological Analysis of Breast Tumor Progression
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Histopathological
analysis protocol of breast tumor refers to the approach described
by Zhao et al.46 (link) with minor modifications.
Tumors from five points in time of progression are frozen into optimal
cutting temperature compound by semiembedding approach and sliced
into 10 μm sections. Tumor sections are stained with hematoxylin
and eosin (H & E) to investigate the histopathological variation
during the tumor progression. The critical dyeing time includes 4
min nuclear stain with hematoxylin and 45-s cytoplasm stain with eosin.
Staining situation is observed by using Leica DM 2500 microscope (Leica,
Germany). Histopathological images are achieved by using a digital
pathology slide scanner (Aperio GT450, Leica, Germany). The morphological
characteristics and areas of glandular cavity, necrosis, and nests
of neoplastic cells are quantified by using Image-Pro Plus software
(Version 6.0, Media cybernetics, USA) at 2× or 10× magnification.
analysis protocol of breast tumor refers to the approach described
by Zhao et al.46 (link) with minor modifications.
Tumors from five points in time of progression are frozen into optimal
cutting temperature compound by semiembedding approach and sliced
into 10 μm sections. Tumor sections are stained with hematoxylin
and eosin (H & E) to investigate the histopathological variation
during the tumor progression. The critical dyeing time includes 4
min nuclear stain with hematoxylin and 45-s cytoplasm stain with eosin.
Staining situation is observed by using Leica DM 2500 microscope (Leica,
Germany). Histopathological images are achieved by using a digital
pathology slide scanner (Aperio GT450, Leica, Germany). The morphological
characteristics and areas of glandular cavity, necrosis, and nests
of neoplastic cells are quantified by using Image-Pro Plus software
(Version 6.0, Media cybernetics, USA) at 2× or 10× magnification.
10
Raman Microspectroscopy Analysis of Collagen Scaffolds
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The specimens of collagen scaffold were cut into 50-μm thick sections using a Leica CM 1950 cryostat (Leica, Germany). The Raman spectra were directly recorded on collagen scaffolds without further preparation by Renishaw (Renishaw Inc., New Mills) via Raman microspectroscopy system equipped with a 300-mW near-infrared diode laser at a wavelength of 785 nm semiconductor laser for excitation. The laser beam of approximately 15 mW was focused on the sample surface by a 50× objective lens of a Leica DM2500 microscope (Leica, Germany). Experiments were performed using a microscope in a backscattering geometry, where the scattering light was collected inside the cone, defined by the objective. The spectra were recorded with a resolution of 1 cm -1 in the range of 700 to 1700 cm -1 and collected with 5 s exposure time and three accumulations. All the data were collected under the same conditions. The raw spectra acquired from the collagen scaffolds in the range of 700 to 1700 cm -1 represented a combination of prominent autofluorescence, weak Raman scattering signals, and noise. Therefore, baseline correction was carried out by using a modified third-order polynomial fitting followed by smoothing.
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