Vibrosections at the cortical level were photographed with the Leica inverse microscope at a 10× magnification under a red filter. The exposure time was always 23 ms with the bright light set at the lowest level. The software Openlab was used at a Mac computer connected to the microscope. Pictures were saved as JPG files and the analysis was performed using Image J. The pictures were transformed to a 8-bit grayscale image. The calibration was set at 0.470 (distance in pixels), 1.00 (known distance), 1.0 (pixel aspect ratio) and μm (unit in lenght) and global was activated. The picture was transformed into a binary image and the threshold was adapted to 30–40. The number of particles was counted setting the size to 100–8000 μm2. The number of plaques was counted in a defined circle in 2.5 mm2 area. Examples of plaque evaluations are given in the Figures 4E–G .
Inverse microscope
Manufactured by Leica
Sourced in Germany
The Leica inverse microscope is a specialized optical instrument designed for examining specimens from the underside. It features a reversed optical path, allowing for the observation of samples that are not suitable for traditional upright microscopes. The core function of the inverse microscope is to provide a clear, magnified view of specimens that are cultured in specialized containers or dishes.
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Lab products found in correlation
6 protocols using inverse microscope
1
Quantifying Cortical Plaques in Microscopy
2
Quantifying DNA Damage in Xenografts
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The level of RKIP (BBI, Shanghai, China) and γH2AX (phospho-S139, Abclonal, Wuhan, China), a marker for DNA double-strand breaks,19 (link) in xenografts were detected by immunohistochemical staining as our previous description. The score of IHC staining was calculated according to the rules based on staining intensity and relative staining area. Absent, weak, moderate, and strong staining was assigned as 0, 1, 2, and 3, respectively. 0%, 0–30%, 30~60%, and > 60% of staining cells were assigned as 0, 1, 2, and 3, respectively. The eventual staining score was the sum of the intensity score and area score. The rate of DNA damaged cells, indicated by γH2AX staining, was obtained by counting the percentage of staining cells in ten random selected microscopic fields under an inverse microscope (Leica, Solms, Germany).
3
ATP Quantification of Cultured Cells
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The cells were seeded into 96-well plates at a density of 30,000 cells/well (100 µL/well), allowed to attach overnight, and incubated with the test substances in triplicate for 2 days. ATP levels were measured by using a luminescence assay as instructed by the manufacturer (Abcam, ab113849). As a control for equal cellular material, phase contrast images of the center of each well were acquired prior to cell lysis using an inverse microscope (Leica) at 50× magnification, and cell density was quantified by using Fiji software. The contents of each well were transferred into an opaque, white 96-well microplate, and luminescence was measured using a multi-mode microplate reader (SpectraMax i3x). After background subtraction, the mean ATP concentration of each group was normalized to the cell density of the corresponding group.
4
Comprehensive H&E Staining Protocol
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H&E staining was performed using H&E Stain Kit (ab245880, Abcam). The tissues were fixed with paraformaldehyde and then embedded in paraffin. The tissue blocks were cut into 5 μm sections using a microtome. For deparaffinization and re-hydration, the sections were subjected to three washes of xylene for 5 min each, two washes of 100% ethanol for 10 min each, two washes of 95% ethanol for 10 min each, and two washes in distilled water for 5 min each. Deparaffinized/hydrated section of the lung tissues was incubated in adequate Mayer’s hematoxylin solution to completely cover tissue section for 5 min. The section was rinsed twice with distilled water to remove excess stain. Then adequate Bluing reagent was applied to cover tissue section for 30 sec. After washing with distilled water, the section was dehydrated in absolute alcohol, followed by staining with Eosin Y solution for 2 min. The section was rinsed using absolute ethanol for three times and then mounted to a slide. The images were collected under an inverse microscope (Leica).
5
Transwell Assay for Cell Invasion
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Transwell assay was carried out as previously described [18 (link)]. Briefly, 2.5 × 104 cells were seeded into a transwell chamber (#3422, Costar, ME, USA), and 750 mL of DMEM medium containing 5% FBS was poured into the lower well. After 20 h, the cells were fixed in methanol and stained with 0.5% crystal violet. After swabbing cells on the upper side of the membrane, the invasive cells on the upper side of the membrane were photographed and counted using an inverse microscope (Leica, Solms, Germany). The experiments were biologically repeated for threes times.
6
Characterization of Avian Splenic Macrophages
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Phase contrast microscopy was used to characterize the development and growth of the primary avian splenic macrophages and to control the cell culture. For that, an inverse microscope (Leica, Bensheim, Germany) was used. Microscopic analysis was performed between 1 and 7 days after seeding the cells into the flasks.
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