Samples were fixed with 4% PFA, washed three times with PBS and stained with toluidine blue (1% in water) for 10 min at RT. The samples were observed using a DM6000B upright microscope and the LAS V 4.0 software (Leica) was used for images acquisition.
Dm6000b upright microscope
Manufactured by Leica
Sourced in Germany
The Leica DM6000B is an upright microscope designed for a wide range of laboratory applications. It features high-resolution optics, a modular design, and advanced illumination systems. The DM6000B is capable of various imaging techniques, providing users with a versatile and reliable tool for their research and analysis needs.
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Lab products found in correlation
Vectashield mounting medium with dapi, by Vector Laboratories (2 mentions)
Las v 4, by Leica (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Methanol free formaldehyde, by Thermo Fisher Scientific (1 mentions)
8 well chamber slide, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rabbit igg h l antibody, by Thermo Fisher Scientific (1 mentions)
Las af 6000, by Leica (1 mentions)
Dfc500 camera, by Leica (1 mentions)
Las x, by Leica (1 mentions)
Dfc290, by Leica (1 mentions)
M205fa combi scope, by Leica (1 mentions)
Orca er, by Hamamatsu Photonics (1 mentions)
Matrigel, by BD (1 mentions)
Transwell insert, by Merck Group (1 mentions)
Technovit 7100, by Kulzer (1 mentions)
Cometslide, by Bio-Techne (1 mentions)
Sybr gold nucleic acid gel stain, by Thermo Fisher Scientific (1 mentions)
Lmagarose, by Bio-Techne (1 mentions)
Lysis buffer, by Bio-Techne (1 mentions)
Cometassay electrophoresis system 2, by Bio-Techne (1 mentions)
13 protocols using dm6000b upright microscope
1
Toluidine Blue Staining of PFA-Fixed Samples
2
Evaluating DNA Damage Response to Small Molecule Treatments
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IEC-6 cells were seeded in 8-well chamber slides (Thermo Fisher Scientific) at 5 × 104 cells per well and serum starved in 2% FBS medium overnight. Cells were treated with DMSO, 3 µM MMC, or the indicated concentration of 742 or 746 for 24 hours before washing once with cold PBS and fixation in 4% methanol free formaldehyde (Thermo Fisher Scientific) for 30 min on ice, washing with PBS and quenching of free aldehydes with a 50 mM NH4Cl solution for 10 minutes. Cells were permeabilized using 0.25% Triton-X 100 (Sigma Aldrich) for 5 min on ice, washed with PBS, and blocked with blocking solution (PBS containing 1% BSA and 5% normal goat serum) for 1 hour on ice and incubated with the Phospho-Histone H2AX (Ser139) rabbit monoclonal antibody (Cell Signaling, 9718) diluted 1:800 in blocking solution overnight at 4°C. Cells were washed with PBS and incubated in the secondary Alexa Fluor 488 goat anti-rabbit IgG (H+L) antibody (Life Technologies, A-11034) for 45 minutes at room temperature. Cells were washed with PBS and mounted with VECTASHIELD mounting medium with DAPI (Vector Labs). Cells were examined for immunofluorescence using a Leica DM6000B upright microscope and counted as positive if more than five foci/nucleus were detected.
3
Isolating and Characterizing Mycobiota Spores from Tunisian Soils
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The mycobiota inoculum was recovered from soils (0- to 20-cm depth) sampled inside a cork oak forest in Tunisia, as previously reported (26 (link)). Each MC3000 cultivation system was inoculated with an inoculum corresponding to 0.5 g of soil. After incubation, the MC3000 filters were immersed in a saline solution (0.9%) containing 0.1% Tween20, to release the adherent spores. Spores on the surface of the cultures were independently collected. Spores were washed (18,000 × g, 20 min, 4°C), resuspended in a 30% glycerol solution, and stored at −80°C. A Neubauer chamber was used to count the number of spores (optical microscope, ×400 magnification), and their sizes were evaluated microscopically upon a calcofluor-white staining (Leica DM 6000B upright microscope, 63 × 1.4 NA oil immersion objective plus a 1.6× Optvar) through analysis of >20 independent microscopic fields per sample (ImageJ and XL-STAT, v1.8.0_172) as follows. The size bar was used to set scale, images were converted to 8-bit format, black-and-white thresholding was used to remove noise, potential holes in the particles were digitally filled, and the particle analysis command used. Results were trimmed (excluding areas <5 μm2) to remove remaining noise, registered, and further analyzed using XL-STAT (Addinsoft, v2014.5.03).
4
Quantifying Ocular Neural Crest Cells
5
Cancer Cell Migration and Invasion
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Migration and invasion experiments were performed as previously described.24 , 25 U87MG and U251 cells (3 × 104 cells/well) were subjected to the upper level of a transwell insert (Millipore, Billerica, USA) with or without matrigel (BD Bioscience, USA). After 24 hours, the membranes were stained with crystal violet and the positive staining cells were randomly counted under a Leica DM6000B upright microscope (Wetzlar, Germany, nine fields, ×400).
6
GUS Staining of Arabidopsis Seedlings
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For Arabidopsis seedlings, the GUS staining was conducted as previously described76 (link). Briefly, the samples were submersed in a GUS staining solution (50 mM sodium phosphate buffer pH 7.0, 0.01% Triton X-100, 1 mM K3Fe(CN)6, 1 mM K4Fe(CN)6, 1 mg.ml−1 5-bromo-4-chloro-3-indolyl β-D -glucuronide) and incubated 1 h at 37 °C. Plants were observed and documented with a DM6000B upright microscope (Leica; × 10 objective) after clearing with chloral hydrate solution as described12 (link).
For histological sections samples were stained for 20–25 min at 37 °C. Samples were fixed (1% glutaraldehyde, 4% paraformaldehyde in 50 mM phosphate buffer, pH 7.0) for 24 h, followed by stepwise dehydration in ethanol. Embedding was accomplished with Technovit 7100 (Kulzer, Germany). Sections (10 μm) were prepared and stained with ruthenium red. Sections were observed and documented with the axio Zoom.V16 (Zeiss) using the × 2.3 objective.
For histological sections samples were stained for 20–25 min at 37 °C. Samples were fixed (1% glutaraldehyde, 4% paraformaldehyde in 50 mM phosphate buffer, pH 7.0) for 24 h, followed by stepwise dehydration in ethanol. Embedding was accomplished with Technovit 7100 (Kulzer, Germany). Sections (10 μm) were prepared and stained with ruthenium red. Sections were observed and documented with the axio Zoom.V16 (Zeiss) using the × 2.3 objective.
7
Comet Assay for Cellular DNA Damage
8
Immunofluorescence Imaging of Catecholaminergic and Astrocytic Markers
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For the catecholaminergic marker tyrosine hydroxylase (TH) and hTyr, immunofluorescent micrographs were acquired on a Leica DM6000B epifluorescent upright microscope at 20x magnification with uniform exposure parameters for each stain and region imaged. Brightfield images of pigment granules were also obtained on the Leica DM6000B upright microscope at 20x magnification. Following convention, these images are oriented with the dorsal direction up and the ventral direction down. For astrocyte marker glial fibrillary acidic protein (GFAP) and LC terminal marker NE transporter (NET), listed in Table 1 , immunofluorescent images were acquired as z-stack images (10 z-stacks; pitch: 0.1 μm) at 20x magnification and compressed on a Keyence BZ-X700 microscope system. One representative atlas-matched section was selected from each animal. Image processing for all images was conducted using the FIJI/ImageJ software.
9
Fluorescence Imaging of Cellular Morphology
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Phase contrast and epifluorescence imaging were performed using a Delta Vision system (Applied Precision Inc.) on an Olympus IX inverted microscope equipped with a cooled CCD camera. For high magnification images a 60×/1.3 NA (Olympus) oil-immersion objective was used. Epifluorescence microscopy was additionally performed using a Leica DM6000B upright microscope equipped with a CCD camera. A water immersion objective 40×/0.8 NA (Leica) was used. Laser scanning confocal microscopy (LCSM) was performed on a Zeiss LSM 5 microscope using a 40×/1.2 NA (Zeiss) water immersion objective.
Cells were fixed with 4% paraformaldehyde in PBS (15 minutes in PBS) and stained with fluorescent agents. Wheat germ agglutinin, AlexaFluo 488 conjugate (WGA; 10 µg/ml) was used to stain plasma membrane, phalloidin-tetramethyl rhodamine B isothiocyanate (Phalloidin-TRITC; 2.5 µg/ml) stained filamentous actin and 4′,6-diamidino-2-phenylindole (DAPI; 1.0 µg/ml) labeled cell nuclei. Projected cell area was calculated using the Cell Outliner plugin of ImageJ software (NIH) from fluorescence microscopy images of WGA-stained cells.
Cells were fixed with 4% paraformaldehyde in PBS (15 minutes in PBS) and stained with fluorescent agents. Wheat germ agglutinin, AlexaFluo 488 conjugate (WGA; 10 µg/ml) was used to stain plasma membrane, phalloidin-tetramethyl rhodamine B isothiocyanate (Phalloidin-TRITC; 2.5 µg/ml) stained filamentous actin and 4′,6-diamidino-2-phenylindole (DAPI; 1.0 µg/ml) labeled cell nuclei. Projected cell area was calculated using the Cell Outliner plugin of ImageJ software (NIH) from fluorescence microscopy images of WGA-stained cells.
10
HMGA2 and MMP2 Immunohistochemistry
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HMGA2 and MMP2 were detected by the VECTASTAIN ABC Detection System (VECTOR, Burlingame, USA) following the manufacturer's instructions. Primary antibodies included mouse anti‐HMGA2 diluted at 1:400 (Abcam, Cambridge, USA) and rabbit anti‐MMP2 diluted at 1:100 (CST, Danvers, USA). The IHC staining was analyzed with a Leica DM6000B upright microscope with CCK digital camera (Wetzlar, Germany). The labeling index [LI (%)] was calculated based on the percentage of cell number between positive staining and total.
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