MCs in cervical lymph node (CLN) or skin tissue was evaluated by toluidine blue staining according to previous report with minor modifications (Huang et al., 2013a (link)). In brief, the tissue (CLN or skin) was immersed in 4% neutral buffered formalin for 48 h before being cut into non-contiguous 5-μm-thick sections (100-μm distance between sections) using a Leica microtome (Leica, Germany). These sections were deparaffinized, rehydrated, immersed in 0.5% toluidine blue (Sigma-Aldrich, USA) for 12 h, and washed in distilled water for 3 min, with three changes. The sections were then dehydrated quickly through 95% ethanol, and two changes of absolute ethanol. The sections were cleared in xylene and mounted with neutral resins. MCs with deep blue-purple staining were analyzed under a Leica DM 2500B microscope. The number of MCs (both intact and degranulated forms) was analyzed by counting three non-contiguous sections (CLN or skin tissue) in each animal (n = 6/group). Meanwhile, the area of section was visualized and measured by using Image-pro Plus software in Leica DM 2500B microscope. The density of MCs (both intact and degranulated forms) in CLN or skin tissue was determined as MCs counts/mm2, and the level of degranulated MCs was calculated as follows: ▲ = degranulated MCs counts/(both intact and degranulated MCs counts) × 100%.
Dm 2500b microscope
Manufactured by Leica
The Leica DM 2500B is a microscope designed for laboratory use. It features a binocular observation tube and a built-in light source. The microscope is capable of bright-field and phase contrast imaging.
Automatically generated - may contain errors
Lab products found in correlation
Dfc420c camera, by Leica (2 mentions)
Dm5500b microscope, by Leica (2 mentions)
Microtome, by Leica (1 mentions)
Toluidine blue, by Merck Group (1 mentions)
Jes fa200 x band spectrometer, by JEOL (1 mentions)
Application suite v4, by Leica (1 mentions)
Ht501128 4l, by Merck Group (1 mentions)
Dfc360 fx camera, by Leica (1 mentions)
Dfc360 fx, by Leica (1 mentions)
8 protocols using dm 2500b microscope
1
Mast Cell Quantification in Lymph Nodes and Skin
2
Electrochemical Characterization of Polymers
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Cyclic voltammograms were obtained using a Bio-Logic SP-150 and CH Instruments Electrochemical Analyzer model 620. A single-cell and three-electrode setup was used (eDAQ Pty Ltd., Denniston East, Australia): 2 mm2 of platinum disk (working electrode), platinum coil (counter electrode), and Ag|Ag+ pseudo-reference electrode calibrated with ferrocene as the internal standard. The sample concentration was 1.0 mM (in the presence of 0.1 M E1 or E2) in DCM. Polymer films were prepared on platinum electrode by electrooxidation after the first oxidation peak. The potential was changed at a rate of 50 mV/s. Argon was bubbled before electrooxidation and electroreduction. ESR spectroelectrochemical measurements were acquired using a JEOL JES FA-200 X-band spectrometer with the following parameters: 0.6 mT (modulation width); 1 mW (microwave power); and 200 (amplitude). A capillary quartz spectroelectrochemical cell was used, equipped with a Pt wire working electrode in monomer solution (1 mM) or with a polymer coating, Ag wire pseudoreference electrode (potential calibrated versus standard potential of Fc|Fc+ couple) and Pt coil as a counter electrode. The surface imaging of electrochemically obtained polymers was performed with Leica DM 2500 B microscope with 50 lenses was used.
3
Semi-Quantitative Evaluation of Implant Wear Particles
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Stained sections were analyzed and photographed by a Leica DM2500b microscope and semiautomatic Leica Application Suite V4 imaging analysis and procedures. Semi-quantitative evaluation of immunohistochemical dye was performed as follows: negative dyeing was defined as (–); the fraction of occasional positive cells (less than 5% of the section area) was defined as (±); some positive cells (5∼25%) were defined as (+); a medium number of positive cells (25∼50%) were defined as (++); and a high number of positive cells (more than 50% of the tissue) were defined as (+++). All the immune-stained tissues were blindly graded and checked through optical microscopy by two different researchers. The evaluations were combined with a histological grading of wear particles. Metal particles were examined microscopically like black stain. Ultrahigh molecular mass-polyethylene particles (UHMMPE) were examined by a polarized optical microscope. Polymethylmethacrylate (PMMA) particles were observed as ‘‘ghosts’’ combined with residually small particles, unsolvable 0.5∼1 μm barium sulfate residues.22 (link)
The extent of the particles were evaluated as follows: (–) absent; (±) occasionally found in the tissues covering under 5% of the area examined; (+) often found in 5–25% of the area; (++) moderately found in 25–50%; (+++),abundantly more than 50%.23 (link)
The extent of the particles were evaluated as follows: (–) absent; (±) occasionally found in the tissues covering under 5% of the area examined; (+) often found in 5–25% of the area; (++) moderately found in 25–50%; (+++),abundantly more than 50%.23 (link)
4
Histopathological Analysis of Inflammatory Foci
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The paraffin-embedded CLN or liver sections (5-μm) were stained with haematoxylin and eosin (H&E) dye, and then analyzed for histopathological changes by two blinded researchers. The inflammatory foci, in three non-contiguous liver sections per animal (n = 6/group), was visualized and analyzed under a Leica DM 2500B microscope at a magnification of × 200. The number of inflammatory foci per field was calculated by counting 10 fields of each section (Cavalcanti et al., 2011 (link)).
5
Histopathological Analysis of Mouse Feet
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Mouse feet used for histopathological analysis were fixed in 10% neutral-buffered formalin solution (4% formaldehyde, Sigma, HT501128-4L) for 24 hours at room temperature, decalcified in 0.6 M EDTA and 0.25 M citric acid for 14 days at 37°C and transferred to 70% EtOH for storage. After dehydration and embedding in paraffin, 5 μm thin sections were cut. Sections were then deparaffinised, rehydrated, and stained with Haematoxylin/Eosin (HE, Sigma, 51275-500ML, J.T. Baker, 3874) or Ziehl-Neelsen/Methylene blue (ZN, Sigma, 21820-1L and 03978-250ML) to stain for mycobacteria according to WHO standard protocols [36 ]. Finally, the sections were mounted with Eukitt mounting medium (Fluka, 03989) and pictures were taken with an Aperio scanner or with a Leica DM2500B microscope.
6
Quantifying Lung Apoptosis in MA-ALI Mice
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TUNEL-DAPI double staining was performed to evaluate lung apoptotic changes in the experimental MA-ALI mice upon GsMTx4 treatment following the manufacturer’s instructions (no. G1501-50 T; Servicebio). Briefly, three non-contiguous lung slices from each animal (n = 4–5/group) were deparaffinized with xylene, rehydrated with graded ethanol solutions and subsequently incubated with 20 mg/ml of proteinase K at 37 °C for 30 min. After rinsing with distilled water, the slices were immersed in TUNEL reaction mixture (CY5-biotinylated dUTP in TdT buffer) at 37 °C for 60 min, restained with 1 μg/ml of DAPI solution at 37 °C for 10 min, coverslipped with anti-fade polyvinylpyrrolidone solution (Beyotime, China) and then examined under a fluorescence microscope. Fluorescent images of TUNEL-positive apoptotic cells with green color and cell nuclei with blue color were captured using a Leica DM 2500B microscope at × 200 magnification. The apoptosis index was determined as apoptosis-positive number/field from more than 20 random fields per lung section (n = 4–5 mice/group).
7
Multidimensional Microscopic Imaging of Cyanobacterial Cells
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A Leica DM2500 B microscope with a Leica DFC420C camera was used for light microscopy. Fluorescence microscopy was performed with a 100x/1.3 oil objective lens of a Leica DM5500 B microscope connected to a Leica DFC360FX camera. GFP and chlorophyll fluorescence were excited using a BP470/40 nm or BP535/50 nm filter and emission was monitored using a BP525/50 nm or BP610/75 nm filter, respectively. Z-stacks with 0.1 µm intervals were taken to perform three-dimensional deconvolution using the built-in function of the Leica ASF software.
For quantification of the fluorescence in the septum, each xy pixel of 20 slices of a raw (not deconvoluted) z-stack was summed into one image and the mean intensity of septal ROIs was measured. Background fluorescence was measured with identical ROIs in the cytoplasm. The averaged background fluorescence was subtracted from every single septum measurement and used for normalization of the values.
To visualize the presence of the heterocyst polysaccharide layer, the cells were stained with Alican blue as described56 .
For quantification of the fluorescence in the septum, each xy pixel of 20 slices of a raw (not deconvoluted) z-stack was summed into one image and the mean intensity of septal ROIs was measured. Background fluorescence was measured with identical ROIs in the cytoplasm. The averaged background fluorescence was subtracted from every single septum measurement and used for normalization of the values.
To visualize the presence of the heterocyst polysaccharide layer, the cells were stained with Alican blue as described56 .
8
Fluorescence Microscopy of Cyanobacterial Heterocysts
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Light microscopy was performed with a Leica DM2500 B microscope connected, for color micrographs, with a Leica DFC420C camera. Images of fluorescence were taken with a Leica DM5500 B microscope with a 100x/1.3 oil objective lens (Leica Microsystems) connected with a Leica DFC360FX black and white camera. For visualization, specific filter cubes for fluorescence microscopy were needed depending on fluorophore spectral properties. GFP was monitored with a BP470 40-nm excitation filter and a BP525 50-nm emission filter. Cyanobacterial auto-fluorescence was monitored using a BP535 50-nm excitation filter and a BP610 75-nm emission filter. Bright-field images were exposed for 10 ms and 80 to 150 ms in the fluorescence channels. Images of fluorescence were re-colored by the Leica ASF software based on the filters used. Filament fragmentation was analyzed as previously described (Merino-Puerto et al., 2010 (link)). To stain the polysaccharide layer of heterocysts, cell suspensions were mixed with a 1% (w/v) solution of Alcian Blue (McKinney, 1953 (link)).
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