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16 protocols using eclipse ci e

1

Evaluating Inflammatory Cytokines in Apical Periodontitis

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All samples were decalcified with a 5.0% aqueous solution of ethylene diamine tetra acetic acid (EDTA) for 90 days, embedded in paraffin blocks, and sliced by microtome at 5 µm thickness following standard histological protocol. Hematoxylin and eosin (HE) staining was used for histopathological examination. Sections containing treated teeth with apical periodontitis were focused on for consideration.
Immunohistochemical (IHC) staining was performed using monoclonal primary antibody anti-IL-6 (ABIN6002527, antibodies-online, Germany) and anti-TNF-a (ABIN3023860, antibodies-online, Germany) to assess macrophage expression. Macrophages were counted in the periapical region. Histopathological and immunohistochemical specimens were analyzed microscopically using a light microscope Nikon Eclipse Ci-E (Nikon Corp, Japan) focusing in the periapical area of apical periodontitis. Expression of IL-6 and TNF-a of macrophages was counted from IHC-stained specimens at 400× magnification.
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2

Lung Micrometastasis Evaluation in Melanoma

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Subcutaneous injection of B16F10 melanoma cells into C57BL/6 mice leads to form primary tumors in 7–9 days and spontaneous micrometastasis in lungs.23 (link) Because macroscopic tumor nodules were not visible at the early stage through subcutaneous injection of B16F10 melanoma cells, hematoxylin and eosin (H&E) staining was used to observe micrometastasis in lungs before and after the cryo-thermal therapy. On day 12, 26 and 102 after tumor inoculation, three mice were randomly selected from each group (on day 102 after tumor inoculation, all mice in the control group were dead) and killed by an intraperitoneal injection of pentobarbital sodium (Sigma-Aldrich). The lung tissues were removed, fixed in 10% formalin, and paraffin-embedded sections were prepared. Paraffin-embedded tissues were sliced into sections of 7 μm each. Hematoxylin and eosin staining of lung tissue was performed and the pathological changes were visualized under a microscope (Nikon Eclipse Ci-E, Nikon, Beijing, China).
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3

Tumor Tissue Analysis in Rats

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Fourteen days after the treatment, four rats were randomly selected from each group and sacrificed by an intrapertoneal injection of barbital sodium (Sigma-Aldrich). The tumors were removed, fixed in 10% formalin and paraffin-embedded sections were prepared. Hematoxylin and eosin staining of the tumor tissue was performed and the pathological changes were visualized under a microscope (Nikon Eclipse Ci-E, Nikon, Beijing, China).
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4

Wound Healing Assay of U87 Glioma Cells

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The U87 glioma cells were transfected with scramble or si-CISD2 plasmid and seeded into a 6-well plate at a confluence of 30% (3×104 cells/well). When cell confluence reached 95%, the cells were starved for 12 h. A 100-ml pipette tip was then used to scratch a straight line in the cell layer. Following incubation for another 24 h, the cells were fixed and images were captured under a microscope (Eclipse Ci-E; Nikon Corporation). The length of the wound was measured using Image J software.
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5

ROS Measurement in Corneal Samples

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ROS production was evaluated based on dihydroethidium (DHE; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) measurements, as previously described (24 (link)). Following removal of the eyeball, the fresh cornea was harvested and immediately flash-frozen in liquid nitrogen (n=4 per group). The frozen sections (10-µm thick) were washed three times with 0.01 M PBS (pH 7.2-7.4) and then incubated with 5 mM DHE dissolved in PBS solution for 30 min at room temperature. DHE specifically reacted with superoxide anion free radicals and was transformed into red fluorescent compounds. The sections were observed and images were captured with a fluorescence microscope (Eclipse Ci-E; Nikon Corporation, Tokyo, Japan) under the same exposure conditions, and the average optical density of the fluorescent dye in the corneal stroma layer was measured using randomly selected images. Three fields of view were observed per animal. The fluorescence intensities of the DHE-tagged cells were quantified with Adobe Photoshop CS5 (Adobe Systems, Inc., Beijing, China).
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6

Measuring Intestinal Morphology in Broilers

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Embedded tissue was deparaffinized and hydrated, cut into slices (5 μm), and then stained with hematoxylin and eosin for morphology measurements as described by Wang et al. (23 (link)). Villus height was measured from the tip of the villus to the crypt–villus junction. Crypt depth was defined as the depth of the invagination between adjacent villi (24 (link)). All morphological measurements (villus height and crypt depth) were measured on the stained sections under a microscope at ×40 combined magnification (Nikon Eclipse Ci-E, Japan). At least 15 intact, well-oriented crypt–villus units were measured in triplicate per broiler for each intestinal section. Reported values are means from 15 crypt–villus units.
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7

Fungal Conidiation Microscopy Protocol

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To observe conidiation processes of different fungal strains, conidial suspensions (100 µL of 1 × 107 conidia/mL) were inoculated on 1/4 SDAY plates and the microcycle conidiation medium (SYA, 0.5% yeast extract, 3% sucrose, 0.05% MgSO4, 0.001% MnSO4, 0.05% KCl, 0.3% NaNO3, 0.1% KH2PO4, 0.001% FeSO4 and 2% agar, w/v), followed by incubation at 28 °C. After 16, 20, 24, 28 and 36 h of cultivations, approximately 1 cm2 was cut for observation using a microscope (Motic, Guangzhou, China). Hyphal samples were stained with 10 µL CFW (50 μg/mL) for 30 min after 18, 24 and 30 h of cultivation to visualize the mycelial septa with a fluorescent microscope (Nikon Eclipse Ci-E, Tokyo, Japan).
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8

Immunohistochemical Analysis of CISD2 in Glioma

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Immunohistochemical staining was performed on the collected clinical glioma tissues sections to detect the levels of CISD2. Briefly, formalin and paraffin were used to fix and embed the tissues, respectively. The samples were heated to retrieve the antigen. The sections were incubated with anti-CISD2 primary antibody (1:500; cat. no. AV44552; Sigma-Aldrich; Merck KGaA) at 4°C overnight. Following three washes with PBS, the sections with incubated with HRP-labeled goat anti-rabbit IgG (1:1,000; cat. no. A0516; Beyotime Institute of Biotechnology) for 2 h. Following three washes with PBS, the sections were visualized with DBA solution, followed by counterstaining of nuclei with hematoxylin. Images were captured using a microscope (Eclipse Ci-E; Nikon, Tokyo, Japan).
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9

Quantifying Hippocampal Neuron Survival via Nissl Staining

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Nissl staining was used to observe neurons in the right hippocampal dentate gyrus (DG) of the insulted hemisphere with Toluidine Blue Staining Reagent (Sigma Aldrich, 89640, USA). Briefly, we dewaxed the paraffin sections (as for TUNEL staining) and stained them with 5% toluidine blue at room temperature for 10 min. Sections were then soaked in 95% ethanol (Sigma Aldrich, 49836, USA) for 2 min, dimethylbenzene (Sigma Aldrich, 296333, USA) for 3 min, and sealed with neutral balsam (Thermo Fisher Scientific Inc., B10100, USA). The Nissl staining slides were observed using a Nikon microscope (Nikon, Eclipse Ci-E, Japan). We used neuronal number per mm2 to measure the survival neurons in Nissl staining. We counted the number of the neurons in 50 × 50 μm2 of the initial images, then calculated the average neuron number per mm2.
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10

Intestinal Histology Analysis via H&E

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About 3 cm of small bowel tissue above 2 cm from the ileocecal area was fixed with 10% formalin, paraffin embedded, and sectioned. After dewaxing with xylene, a series of ethanol was used for hydration. The sections were stained with hematoxylin (B600020; Proteintech, China) for 5 min. After washing with alkaline PBS and bluing for 2 min, they were rinsed with running water for 3 min. Afterwards, they were stained with eosin (Sigma, USA) for 10 min and flash washed with distilled water. Neutral gums were used for coverslipping. The morphological and structural changes of intestinal mucosal epithelium were observed under an optical microscope (eclipse Ci-e; Nikon, Japan). According to the results of hematoxylin and eosin staining, the degree of intestinal injury was evaluated by Chiu's score [15 (link)].
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