All photothrombic strokes were induced using this protocol as previously described (Zheng et al., 2010 (link), 2013 (link)), except for the data presented in Figures 5A ,C . Briefly, mice were anesthetized with 4% isoflurane and maintained at 2% isoflurane throughout the surgery. Hair was removed, and incision made on the dorsal scalp, and head mounted in a custom frame. Either a cranial window or a thin skull prep was performed. Rose Bengal (Sigma, Cat no 330000) dye was then injected intravenously, and a blood clot induced with a 568 nm laser on a Nikon (TE 200) microscope, with blood vessels between 30 and 40μM targeted for clotting. Mice were injected with drugs [MRS2365: Tocris Cat no 2157; MRS1523: Sigma Cat no M1809; Fluoroacetate: Sigma Cat no 62-74-8; MRS5698: Tocris Cat no 5428; Cl-IB-MECA: Tocris Cat no 1104; AST-004, synthesized at the National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, MD (Ravi et al., 2002 (link))] either before surgery or 30 min post-stroke, as described in the paper.
Te200 microscope
Manufactured by Nikon
Sourced in Japan, United States
The Nikon TE200 microscope is a laboratory instrument designed for high-performance optical imaging. It features a stable, ergonomic design and offers a range of advanced optics for detailed observation and analysis. The TE200 provides consistent and reliable performance for various microscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
Harris s hematoxylin, by Merck Group (2 mentions)
Masson s trichrome, by Merck Group (2 mentions)
Metamorph software, by Universal Imaging (2 mentions)
Ds u2 pc control unit, by Nikon (2 mentions)
Nis element microscope imaging software, by National Instruments (2 mentions)
Ds fi1c camera, by Nikon (2 mentions)
Rose bengal, by Merck Group (1 mentions)
Mrs2365, by Bio-Techne (1 mentions)
Mrs1523, by Merck Group (1 mentions)
Fluoroacetate, by Merck Group (1 mentions)
Mrs5698, by Bio-Techne (1 mentions)
Cl ib meca, by Bio-Techne (1 mentions)
Te200 inverted microscope, by Nikon (1 mentions)
Aqueous permanent mounting medium, by Agilent Technologies (1 mentions)
Gomori trichrome, by Merck Group (1 mentions)
Eosin y, by Merck Group (1 mentions)
Hematoxylin and eosin, by Merck Group (1 mentions)
Nis elements software, by Nikon (1 mentions)
Transwell with 8.0 μm pore polycarbonate membrane insert, by Corning (1 mentions)
Ab30788, by Abcam (1 mentions)
27 protocols using te200 microscope
1
Photothrombic Stroke Mouse Model
2
Soft Agar Clonogenic Assay for EVs
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Cells were harvested for soft agar cloning after a 7 days co-culture with EVs. Soft agar cloning was examined using 0.7 % agarose in PBS and mixed with 1× media with 15 % FBS. The top layer consisted of 0.35 % agarose in PBS, 1× media with 22.5 % FBS and 1 x 105 cells per dish. Observations were made under the 40x objective and images of the plates were captured by a Nikon TE200 microscope.
Gray-scale Images (8 bit) were acquired with a Nikon TE200 inverted microscope (Nikon Inc. Melville NY) using a 10X Plan Fluor objective. Images were captured with a Spot RT3 digital camera (Diagnostic instruments, Sterling Heights MI) using the cameras built-in green filter to increase image contrast. iVision image analysis software (BioVision Technologies version 4.5.4, Exton, PA) was used to calculate area of the colonies. Images were calibrated so area measurements are expressed in micrometers.
Gray-scale Images (8 bit) were acquired with a Nikon TE200 inverted microscope (Nikon Inc. Melville NY) using a 10X Plan Fluor objective. Images were captured with a Spot RT3 digital camera (Diagnostic instruments, Sterling Heights MI) using the cameras built-in green filter to increase image contrast. iVision image analysis software (BioVision Technologies version 4.5.4, Exton, PA) was used to calculate area of the colonies. Images were calibrated so area measurements are expressed in micrometers.
3
Teratoma Cryosectioning and Staining
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The 10 μm thick sections were collected from frozen teratomas using a cryostat (Microm HM 505 N; Microm International GmbH, Dreieich, Hessen, Germany), air-dried, stained for 10 min with Harris’s hematoxylin (Sigma-Aldrich) and 40 min with Gomori trichrome (Sigma-Aldrich). Finally, sections were mounted in aqueous permanent mounting medium (Dako, Carpinteria, CA, USA). Pictures were taken using a Nikon TE200 microscope (Nikon Instruments, Tokyo, Japan) and NIS Elements software.
4
Live-cell imaging of neutrophil transmigration
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The live-cell fluorescence microscopy flow model has been described previously41 (link). HUVEC monolayers on coverslips were activated with TNF-α (10 ng ml−1, 24 h) and inserted into the flow chamber. Neutrophils (1 × 106 per ml) were drawn across HUVECs at 1.0 dynes per cm2 and adhesion and transmigration monitored by time-lapse, live-cell microscopy performed on a Nikon-TE-200 microscope equipped with a × 20 objective and a heating stage maintained at 37 °C. Differential interference contrast (DIC) optics was used to visualize neutrophil transmigration.
5
Histological Analysis of Teratoma Sections
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Using a cryostat, 10-μm-thick sections were obtained from frozen teratomas (Microm HM 505N; Microm International GmbH), air-dried, stained with Harris's hematoxylin (Sigma–Aldrich) and eosin Y (Sigma–Aldrich) for 7 min, and mounted in aqueous permanent mounting agent for microscopy (Dako). Paraplast sections were stained with Harris's hematoxylin, Masson's Trichrome (Sigma–Aldrich), or Harris's hematoxylin and Gomori Trichrome (Sigma–Aldrich) according to the manufacturer's instructions. Sections were analyzed using a Nikon TE200 microscope (Nikon Instruments) and NIS Elements software.
6
Histological Analysis of Frozen Mouse Muscles
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The frozen mouse muscles were cut into 10 μm sections using cryostat (Microm HM505N, MICROM International GmbH, Walldorf, Germany). The cross sections were placed on slides and, after drying at room temperature, stored at 4 °C for further analyzes. For histological analysis, the sections were hydrated in PBS (10 min) and then stained with hematoxylin and eosin (Sigma Aldrich) or Masson’s Trichrome (Sigma Aldrich), according to the manufacturer’s instructions. The stained sections of each muscle were analyzed using a Nikon TE200 microscope and NIS Elements software (Nikon, Minato City, Tokyo). The area occupied by connective tissue in relation to the area of the entire section was determined using ImageJ 1.8.0 software (National Institutes of Health, Bethesda, MD, USA). For each experiment at least 20 sections were analyzed.
7
Transwell Migration Assay Protocol
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Cells were plated at a density of 6 × 104 cells in the upper chamber of a 6.5-mm Transwell with 8.0 μm pore polycarbonate membrane inserts (Corning). One hundred microliters of serum-free DMEM was added to the Transwells for 8 hours at 37 °C. Complete growth medium was placed in the bottom section as a chemoattractant. Non-migrated cells were wiped off the upper surface using cotton swabs. Cells migrating to the lower surface were fixed and stained using 0.5% crystal violet, imaged using a 10X objective on a Nikon TE200 microscope, and quantified using Image J analysis software.
8
Confocal Raman Microscopy for Single-Cell Analysis
9
Quantifying Protein Dynamics via FRAP
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FRAP was performed on a Nikon TE200 microscope with a 100× NA 1.45 objective. Twenty pre-bleach images were acquired at an exposure time of 100 msec. The GFP signals at the TS, nuclear speckles and nucleoplasm were simultaneously bleached at 488 nm in a region of interest (ROI) 1 µm in diameter at a 56% laser power for 300 msec, and 300 post-bleach frames were collected every 100 msec for 20 sec. Signal recovery was recorded at ≤1% of the power 488-nm laser line using MetaMorph software (Universal Imaging Corp.). Images were analyzed by recording the fluorescence of the bleached region. The background was removed, the intensities at each time point were corrected for bleaching by dividing them by the total cell fluorescence, and these values were finally normalized by dividing them by the fluorescent intensity before bleaching. Half-time values were obtained from recovery curves in which the first pre-bleach value was normalized to 0. The IFs were calculated according to the following equation: 1 − IE where 1 is the total amount of the protein and IE represents the end value of the recovered fluorescence intensity. The data shown in Table 1 correspond to the mean ± SEM of individual cells. P values were calculated using a non-parametric Mann–Whitney test.
10
Somatostatin Immunohistochemistry in DRGs
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Pieces of fresh frozen DRGs were placed in Tissue-Tek (Sakura, 4583) and sectioned to 10-14 µm thickness. Sections were placed onto Superfrost Plus Gold Microscope slides (Fisher, 15-188-48). Following sectioning, slides were placed in a 4% PFA (Sigma, St. Louis, MO, USA, P6148) for 10 min, washed with 1X PBS 3 times, and then stored at room temperature for 60 min with a 5% BSA (VWR, 0332-100G; w/v), 0.1% Triton-X100 (Sigma, T8787; v/v) blocking solution. The slides were then washed in 1X PBS 3 more times and then stored overnight at 4°C with the primary antibody in a 5% BSA, 0.1% Triton-X100 solution. The following day, slides were washed 3 times in 1X PBS and then incubated at room temperature for 60 min in a 2% BSA, 0.01% Triton-X100 solution with the secondary antibody. Slides were then washed 3 times in 1X PBS, vacuum dried, and mounted with Prolong Diamond Antifade mountant with DAPI (Thermofisher, P36962). Slides were stored at 4°C in the dark until imaging on a Nikon TE200 microscope. The following primary antibody was used to stain for SST: rat anti-human SST (abcam, ab30788; 1:500, which was selected because human and dog SST are 98% identical according to an alignment run using NCBI's BLASTp program. The following secondary antibody was used at a 1:200 dilution goat anti-rat IgG conjugated to Cy3 (Life Technologies, Waltham, MA, USA, A10522).
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