The human corneal epithelial cells on eight-chamber slides were fixed with 100% methanol at 4 °C for 10 min and permeabilized with 0.2% Triton X-100 in PBS (1X; 150 mM sodium chloride, 10 mM sodium phosphate, pH 7.3) at room temperature for 10 min. Primary goat polyclonal antibody (Ab) against human MMP-13, MMP-3, or MMP-7 (dilution 1:50) were used for immunofluorescent staining. Alexa-Fluor 488 conjugated secondary antibodies were applied, and propidium iodide (PI) was used for nuclear counterstaining. Secondary antibody alone without primary antibody was used as negative control. The results were photographed with epifluorescence microscopy (Eclipes 400; Nikon, Garden City, NY) using a digital camera (DMX1200, Nikon).
Dmx1200
Manufactured by Nikon
Sourced in Japan, United States, Italy
The DMX1200 is a digital X-ray microscope designed for high-resolution imaging. It features a compact design and advanced imaging capabilities for a wide range of applications in materials science, life sciences, and related fields. The DMX1200 provides detailed, non-destructive analysis of samples at the micro and nanoscale.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse e600, by Nikon (6 mentions)
Act 1 v2, by Nikon (5 mentions)
Eclipse 400, by Nikon (3 mentions)
Eclipes 400, by Nikon (2 mentions)
Faramount mounting medium, by Agilent Technologies (2 mentions)
Oil red o, by Merck Group (2 mentions)
Axioplan, by Zeiss (2 mentions)
Act 1 acquisition software, by Nikon (2 mentions)
Lucia g 4, by Laboratory Imaging (2 mentions)
Eclipse microscope, by Nikon (1 mentions)
Illustrator cs5, by Adobe (1 mentions)
Mach 1, by Biocare Medical (1 mentions)
Sc 12732, by Santa Cruz Biotechnology (1 mentions)
Axioplan 2, by Zeiss (1 mentions)
Fluoview 1000, by Zeiss (1 mentions)
Axioplan 2, by Nikon (1 mentions)
Orca flash 4.0 scmos camera, by Hamamatsu Photonics (1 mentions)
Eclipse e800, by Nikon (1 mentions)
Eclipse 80i light microscope, by Nikon (1 mentions)
Eclipse ts2 microscope, by Nikon (1 mentions)
32 protocols using dmx1200
1
Immunofluorescent Staining of MMP Proteins
2
Anatomical Tracing of Thalamo-Cortical Connections
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These mounted and coverslipped sections were used for light microscopic analysis and cytoarchitectonic localization of the injection site in the thalamus and the anterogradely labeled axons in the cortex. Sections were imaged with a Nikon Eclipse microscope equipped with a Nikon DMX1200 camera. Axonal labeling was sharp and complete, allowing the measurement of structures near the edge of optical resolution such as varicosities and interbouton distances. Varicosity sizes (maximal projection areas) and interbouton distances (from the center of 2 consecutives boutons) were measured on the images using the polyline and polygon tools of the NIS-Elements v3.2 imaging software (Nikon, Tokyo, Japan).
In each experiment, the location and extent of the BDA deposit was reconstructed on the serial coronal sections with the aid of the thionin counterstain. Dark-field illumination and the Franklin and Paxinos atlas were used to delineate thalamic nuclei. Out of the 12 BDA injected hemispheres, 4 showed BDA injections restricted to the vibrissal domain of VPMdm (Fig.1 A, Supplementary Fig. 1 ) and heavy axon labeling confined to E8-9/D8 S1 barrels in S1 (Fig. 1 B,C).
In each experiment, the location and extent of the BDA deposit was reconstructed on the serial coronal sections with the aid of the thionin counterstain. Dark-field illumination and the Franklin and Paxinos atlas were used to delineate thalamic nuclei. Out of the 12 BDA injected hemispheres, 4 showed BDA injections restricted to the vibrissal domain of VPMdm (Fig.
3
Immunopositive Area Quantification in Microscopy
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Slices processed for IHC (lectin and GFAP) were visualized with a Nikon Eclipse E600 microscope. Images were acquired at 10× magnification in bright field using a Nikon DMX 1200 digital camera and ACT-1 acquisition software (Nikon). Pictures of matched areas in each slice (selected as described previously) (29 (link)) were taken and the immunopositive area in each picture (expressed in pixels) was calculated using the ImageJ software. Tissue slices from UT WT mice were used to set the signal threshold.
4
Epifluorescence Microscopy for Tissue Analysis
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The sections were observed and photographed on an E600 epifluorescence microscope equipped with a DMX1200 digital camera driven by the Automatic Camera Tamer software (Nikon Canada, Mississauga, ON, Canada). Photoshop CS5 software (Adobe Systems, San Jose, CA) was used to merge the photomicrographs. Photomicrographs taken with a 20X objective were assembled with the Photomerge function in Photoshop CS5. The sections were then drawn from the photomontage. The outline of the sections and OB glomeruli (GSIB4 labeling), as well as DA+ processes and cells were then drawn with precision on Illustrator CS5 software (Adobe Systems). The accuracy of the illustrations was validated under the microscope. The red labeling was transformed to magenta in Photoshop CS5. The only other changes made to the images were brightness and contrast adjustments.
5
Immunofluorescence Analysis of HLA-E Expression
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HLA-E expression was analyzed by immunofluorescence with anti-HLA-E-PE antibody (MEM-E/08-PE), as previously described [20 (link),21 (link)]. In particular, 105 cells were spotted onto glass slides and fixed by cold acetone for 30 min at 20 °C. Slides were air-dried and kept at 20 °C until use. For assay, slides were rehydrated by washings in PBS, and incubated with anti HLA-E-PE-labelled antibody diluted 1:100 in PBS for 35 min at 37 °C in a humidified chamber. Slides were washed twice with PBS for 10 min, once for 1 min with tap water, and once for 1 min with distilled water. After washings as described, slides were stained with 1:2000 in PBS diluted Hoechst stock solution (ThermoFisher, Milano, Italy) for 5–10 min, washed in distilled water, and finally mounted with 50% glycerol in PBS for fluorescence microscope observation. All samples were observed under a UV light microscope (Nikon Eclipse E600) equipped with a digital camera (DMX 1200).
6
Immunohistochemical Analysis of Tissue Samples
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Immunohistochemical stains were performed on selected sections using horseradish peroxidase (HRP) method. Antigen retrieval pretreatments were performed using a HIER (Heat-Induced Epitope Retrieval) citrate buffer pH 6.0 (Bio-Optica, Milan) for 20 min at 98°C. Primary antibodies were mouse monoclonal cytokeratin (Clone AE1/AE3, DAKO Italia, Milan), mouse monoclonal vimentin (Clone V9, DAKO Italia, Milan), monoclonal mouse CD3 (Clone F7.2.38), mouse monoclonal CD79acy (Clone HM57, DAKO Italia, Milan) and mouse monoclonal b-amyloid 1-16 (Clone 6E10, Covance Italy, Rome). Working solution were 1:100 for cytokeratin, 1:100 for vimentin, 1:50 for CD3, 1:50 for CD79acy and 1:100 for b-amyloid 1-16. Antigen-antibody binding were detected by a HRP polymer detection kit (MACH1, BioCare, Italy) and the slides were counterstained with haematoxylin.
Histological specimens were examined and photographed with a light microscope (Nikon eclipse E600) associated to a microphotography system (Nikon digital camera DMX1200).
Histological specimens were examined and photographed with a light microscope (Nikon eclipse E600) associated to a microphotography system (Nikon digital camera DMX1200).
7
Quantifying Liver Inflammation Markers
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The left lobes of livers were harvested and four equal pieces were snap frozen in liquid nitrogen for further analyses. Frozen liver sections (7 μm) were stained for the presence of infiltrating macrophages and neutrophils as described previously (Bieghs et al., 2012a (link)). For the presence of neutrophils, sections were stained with mouse anti-Ly6G (1:50; BD PharMingen). Cell nuclei were counterstained with hematoxylin (Klinipath). Pictures were taken with a Nikon digital camera DMX1200 and ACT-1 v2.63 software (Nikon). The number of positive-stained cells was counted in six pictures (200 ×) per liver per mouse to determine the level of liver inflammation.
8
Evaluating Myogenin Expression in E17.5 Mice
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To evaluate the expression of myogenin in E17.5 mice, 4 μm sections were deparaffinized in xylene and rehydrated in a decreasing series of alcohol. Peroxidases were blocked with a solution of hydrogen peroxide and methanol (4:1) for 15 min. Antigen retrieval pretreatments were performed using a HIER citrate buffer, pH 6.0 (Bio-Optica, Milan, Italy) for 20 min at 98 °C. We used a mouse monoclonal antibody anti-myogenin (sc-12732; Santa Cruz Biotechnology, Dallas, TX, USA) at 1:50 in PBS. The antibody has a mouse host; for this reason, immunohistochemistry was performed according to the protocol indicated by the M.O.M. Kit (Cat. No. PK-2200, Vector Laboratories, Burlingame, CA, USA) [31 (link)].
The DAB stain was quantified with FIJI ImageJ. For each case, three random 40× fields of the transverse section of the limb skeletal muscle were photographed under an optical microscope (Nikon E600; Nikon, Tokyo, Japan) associated with a digital camera (Nikon DMX1200).
The DAB stain was quantified with FIJI ImageJ. For each case, three random 40× fields of the transverse section of the limb skeletal muscle were photographed under an optical microscope (Nikon E600; Nikon, Tokyo, Japan) associated with a digital camera (Nikon DMX1200).
9
Imaging Cilia Dynamics in Zebrafish
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Immunofluorescence images were captured on an Olympus Fluoview 1000 or a Zeiss Apotome Axiovert Z1 microscope with 60×, 1.4 NA oil immersion objectives. Bright-field images were captured with a Zeiss Axioplan 2 compound microscope using a Nikon DMX1200 digital camera. Live imaging of cilia were performed in Tg(actinb2::Arl13b-GFP) embryos or in embryos injected with 188 pg of RNA encoding Arl13b-GFP (Borovina et al., 2010 (link)) using a Zeiss Axioplan 2 compound microscope fitted with either an Evolve 512 EMCCD camera (Photometrics) or an Orca Flash 4.0 sCMOS camera (Hamamatsu). Images were processed in Adobe Photoshop. Cilia length measurements and movie editing were performed using ImageJ. Figures were assembled using Adobe Illustrator.
10
Hypoxia Inhibition in Tumor Xenografts
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All animal experiments were carried out in accordance with UK Home Office regulations. FaDu cells bearing a HRE-luc reporter (Cignal Lenti HIF Reporter luciferase (SABiosciences)), for hypoxia were described and validated previously [38] (link). 1 × 106 cells were inoculated subcutaneously in athymic nude mice. Mice were divided into cohorts of n = 4 mice per cohort. Drug administration by oral gavage was initiated once tumors reached 100 mm3 volume, as determined using calipers. Tumors were treated for 7 days with BEZ235 at 0, 20 or 30 mg/kg in NMP/PEG300 (Novartis Pharmaceuticals, Basel, Switzerland). Decreases in hypoxia were seen at 3 days with maximal effects by 5–7 days. We failed to observe effects at earlier times. Thus we used 7 days for these experiments. Imaging of hypoxia and perfusion was carried out as previously described [38] (link). Before sacrifice EF5 was administered as previously described [38] (link). Tumor tissue was fixed and stained for EF5 and Ki-67. Images were acquired using a Nikon Eclipse E800 microscope with a Nikon DMX1200 digital camera and measured with ImageJ.
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