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33 protocols using dm2000 led microscope

1

Morphological Analysis of Acuariid Nematodes

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For morphological and morphometric analysis, five specimens were mounted in lactophenol, placed on glass slides, and covered with coverslips. They were assessed for their structures using a Leica® (Wetzlar, Germany) DM2000 Led microscope coupled to a digital camera Leica® ICC50 W, with the support of LAS V4.13 software, and photos of important structures were taken. The helminths were identified at the genus level using determination keys [10 (link),11 ] and other studies for further details [2 (link),8 (link),12 (link)]. The acuariid nematodes of terrestrial birds, subfamily Acuariinae, are characterized by cephalic ornamentation, consisting of cordons extending longitudinally and expanding on the cervical region. The cordons of the specimens were long, not enlarged posteriorly, recurrent, and anastomosing [11 ].
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2

Immunohistochemical Detection of HIF-1α

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The arsenic chronically exposed cells as indicated were grown on sterile chamber slides (Thermo Fisher Scientific) overnight at 37 ºC. Cells were fixed in 3.7% formaldehyde for 10 min, permeabilized in 0.2% Triton X-100 for 10 min and blocked in 5% BSA for 1 h at 25 °C. The HIF-1α antibody (H1alpha67, Abcam, Cambridge, MA, USA) was used at 1 : 200 dilution in 5% BSA containing 0.2% Triton X-100 at 4 °C overnight. An HRP-conjugated polymer-based detection reagent for mouse IgG (Cell Signaling Technology) was used as the secondary antibody at room temperature for 30 min. Reactions were visualized with DAB (Vector Laboratories, Burlingame, CA, USA) and the images were captured by Leica DM 2000 LED microscope (Buffalo Grove, IL, USA).
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3

In vitro Platelet Chemotaxis Assay

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Inflammatory platelet function downstream of purinergic receptor activation by ADP was also investigated through in vitro platelet chemotaxis as previously described, but with minor amendments (29 (link)). Washed platelets (5 × 107/mL) were treated with 2-mM CaCl2 before stimulation with vehicle (PBS) or ADP for 5 min at room temperature. Platelets were then added to the top insert of the transwell plate, with the chemoattractant in the bottom well (0/30 nM fMLP in RPMI 1640 cell media). After 90-min incubation at 37°C, media from the bottom chamber was stained with Stromatol (1:0.5) and platelets were quantified using an improved Neubauer hemocytometer and a Leica DM 2000 LED microscope with an 40× objective lens.
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4

Amyloid-Beta Immunohistochemistry in Mouse Brain

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Immediately following euthanasia, the brain was removed and immersion fixed in 10% buffered formal saline (Pioneer Research Chemicals, UK). After 48-72 h, the brain was blocked into 3 mm coronal slices using an adult mouse brain matrix, and slices were embedded in paraffin wax using a Sakura VIP6 Automated Vacuum Tissue Processor. A series of 4 μm sections comprising the dorsal hippocampus were cut and mounted onto SuperFrost Plus glass slides. For Aβ immunostaining, sections were dewaxed, rehydrated through an alcohol series to water, pre-treated with 80% formic acid for 8 min, followed by washing in distilled water for 5 min. Sections were loaded as wet mounts into a Ventana Discovery XT automated stainer, where further pre-treatment for 30 min with mild CC1 (EDTA boric acid buffer, pH 9.0) and blocking for 8 min with Superblock (Medite, #88-4101-00) were performed prior to incubation for 8 h with biotinylated mouse monoclonal antibody 4G8 (2 μg/ml; Sigma-Aldrich SIG-39240 Beta-Amyloid). Staining was completed with a Ventana XT DABMap kit and a Haematoxylin counterstain, followed by dehydration and permanent mounting with DPX. All images were acquired using a Leica DM2000 LED microscope fitted with a MC190 HD camera.
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5

Yeast-Fungus Interaction Evaluation

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The determination of the interaction between selected yeasts and P. glabrum isolates was evaluated on PDA plates (8 mL per plate) according to Ruiz-Moyano et al. [53 (link)]. Samples of 5 μL of conidia suspensions were inoculated in the middle of the plates. Then, 5 μL of yeast cell suspensions (106 cells/mL) were inoculated at the border of the mold inocula. The interaction was observed after 12, 24, and 48 h of incubation at 25 °C. Then the cultures were washed under tap water for 60 s, and the plates were observed with a Leica DM 2000 LED microscope.
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6

Quantitative Ki67 Immunohistochemistry Analysis

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Xenograft tumours were fixed overnight in 4% paraformaldehyde, before dehydrating and embedding in paraffin. 5 µm sections were stained with H&E (Sigma). Antigen retrieval was carried out in citrate buffer (S236984, Dako) and endogenous peroxidases were blocked with 3% H2O2 in methanol (0390D, Fisher Chemical). Sections were incubated with anti-Ki67 (ab15580, Abcam) primary antibody and stained with biotin-conjugated secondary antibody followed by streptavidin-HRP-based DAB substrate development (K3468, Dako). Images were acquired with the 40x objective on a Leica DM2000 LED microscope and processed with the LAS-X software. Images were evaluated by Fiji (ImageJ). Error bars of figures represent SD after error propagation. For statistical testing, a paired two-tailed Student’s t-test was performed at the 5% significance level.
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7

Platelet Chemotaxis Downstream of Purinergic Signaling

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Inflammatory platelet function downstream of purinergic receptor activation induced by endogenous nucleotide agonists was also investigated through in vitro platelet chemotaxis, as previously described, but with minor amendments (Amison, Jamshidi, et al., 2018 (link)). Washed platelets (5 × 107 mL−1) were treated with 2‐mM CaCl2 before stimulation with vehicle (PBS) or individual agonists for 5 min at room temperature. In some studies, platelets were incubated with antagonists for 10 min at room temperature, prior to agonist stimulation. Platelets (80 μL) were then added to the top insert of the 96‐well Transwell plate, with chemoattractant in the bottom well (0/30 nM fMLP in RPMI 1640 cell media). Following 90‐min incubation at 37°C, media from the bottom chamber was stained with Stromatol (1:0.5) and platelets were quantified using an Improved Neubauer haemocytometer and a Leica DM 2000 LED microscope with an ×40 objective lens.
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8

Immunohistochemical Analysis of EFHD2 and ABCC1 in Lung Adenocarcinoma Xenografts

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H1299 lung adenocarcinoma paraffin sections from mouse xenograft tumors were deparaffinized, hydrated, and heated to 95–100 °C to induce antigen retrieval. After inactivating endogenous peroxidase activity, rabbit anti-human EFHD2 and ABCC1 polyclonal antibodies were used for IHC staining, which was performed by an automatic BenchMark XT staining machine using iVIEW 3,3-diaminobenzidine (DAB) detection kit (Ventana Medical Systems). Tumor sections were finally incubated with iVIEW copper to enhance signal intensity. Samples were then counterstained with hematoxylin, dehydrated, mounted, and examined using a Leica DM2000 LED microscope.
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9

Microscopic Imaging of S. venezuelae

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Coverslips were placed on the surface of S. venezuelae colony and put on a thin agarose pad on a microscope slide for microscopy. Cells were imaged using the Leica DM2000 LED microscope at 100× magnification. Digital images were organized using ADOBE photoshop software.
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10

Helminth Detection Techniques

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Formal‐ether sedimentation and sheathers sugar floatation techniques (Lee et al., 2010) were used for microscopic helminth detection. In both approaches, the slides were examined at 400X in duplicates in a Leica DM2000 LED microscope equipped with a digital camera control unit (Leica DFC 450) and representative images captured. Parasites were identified on the basis of egg colour, shape, internal contents and larvae according to (Kouassi et al., 2015).
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