After fixation of skin tissues with 4% formaldehyde, tissues were embedded in paraffin blocks, cut and mounted on glass slides, slides were deparaffinized and hydrated, and then permeabilized in 300 μl of 0.5% Triton X-100. For immunostaining, the primary antibody was added and then the Alexa Fluor 488-labeled secondary antibody was added. The 4,6-diamidino-2-phenylindole (DAPI) solution was added for nuclei staining of the cells on the skin tissues. Fluorescence-labeled proteins were analyzed using the Nikon Eclipse TE2000-E Confocal microscope with EZ-C1 software (Nikon Corporation, Minato-ku, Tokyo, JPN).
Eclipse te2000 e confocal microscope
Manufactured by Nikon
Sourced in Japan
The Nikon Eclipse TE2000-E Confocal microscope is a laboratory instrument designed for high-resolution imaging of biological samples. It utilizes confocal scanning technology to capture detailed, optical sections of specimens with increased contrast and resolution compared to conventional microscopes.
Automatically generated - may contain errors
Lab products found in correlation
Ez c1, by Nikon (2 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (2 mentions)
Effectene transfection reagent kit, by Qiagen (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Ez c1 software program, by Nikon (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
Matrigel, by BD (1 mentions)
Collagen 1, by BD (1 mentions)
Cellmask green plasma membrane stain, by Thermo Fisher Scientific (1 mentions)
Alexa flour 488, by Thermo Fisher Scientific (1 mentions)
10 protocols using eclipse te2000 e confocal microscope
1
Immunostaining of Skin Tissues
2
Immunostaining of Skin Tissues
3
Subcellular Localization of BmADARa in Sf9 Cells
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To observe the subcellular localization of BmADARa directly, Sf9 cells were removed from 75% ethanol using a coverslip and gently placed in a 6-well culture plate. Irradiation was carried out for 2–3 h at a distance of 20–30 cm from the direct range of the ultraviolet lamp, after which the cells were grown overnight on a glass sheet for confocal microscopy (Olympus, Tokyo, Japan). When 80% of the Sf9 cells converged, the cells were inoculated into 6-well cell culture cluster (Beaver) (2 × 105 cells per well) for the transient transfection. The plate was incubated at 37 °C for 48 h in a 5% CO2 water bath incubator. When the adherent cells were grown to cover 2/3 of the bottom of the plate, the medium was removed and 2 µg of BmADARa-pIZ-EGFP plasmid was transfected into the corresponding well by an Effectene Transfection Reagent Kit (QIAGEN, Dusseldorf, Germany). The transfected cells were washed, fixed and stained according to the method of Yu et al. [27 (link)], and then photographed using a Nikon Eclipse TE 2000-E Confocal Microscope (Nikon, Japan). Each image shown is a representative example of n ≥ 5. Transfection of pIZ/V5-EGFP was used as a control.
4
Visualizing hGnF Adhesion to Membranes
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The adhesion of hGnFs to membranes 2 h after seeding was observed by immunofluorescence imaging. First, hGnFs were seeded at a density of 2000 cells per membrane in 48-well plates. After 2 h, the cells were washed twice with phosphate buffered saline (PBS) and fixed in 3.7% formaldehyde solution in PBS for 20 min at room temperature. Cells were then washed three times with PBS, incubated in PBS containing 0.1% TritonX-100 (Sigma, St. Louis, MO, USA) for 5 min, washed three times with PBS, mounted on slide glasses on membranes, and incubated with 100 nM rhodamine phalloidin (Invitrogen, Corp., Carlsbad, CA, USA) for 30 min. After washing with PBS, cells were incubated with 10 μg/mL 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, Corp., Carlsbad, CA, USA) for 10 min. Images were acquired using a Nikon Eclipse TE2000-E confocal microscope (Nikon, Tokyo, Japan).
5
Immunohistochemical Analysis of Mouse Skin
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The souse skin tissues were fixed in 4% formaldehyde and then embedded in paraffin blocks before being cut into 4 µm slices and mounted onto slides for analysis. The slides with mouse skin tissues were deparaffinized and hydrated and then permeabilized in 0.5% Triton X-100. The mouse skin tissues were incubated with anti-COX-2, anti-keratin 17, or anti-phospho-EGFR (Tyr1068), and then exposed to an Alexa Fluor 488-labeled secondary antibody. For nuclei staining, the cells were incubated with Fluoro-Gel II with DAPI (4,6-diamidino-2-phenylindole) solution (Electron Microscopy Sciences, Hatfield, PA, USA). The cell images were then analyzed using the Nikon Eclipse TE2000-E confocal microscope equipped with the EZ-C1 software program (Nikon Corporation, Minato-ku, Tokyo, Japan).
6
Immunohistochemistry of Ovarian Samples
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Immunohistochemical staining of ovarian samples was performed using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA) in accordance with the manufacturer’s instructions. The samples were fixed in 10% neutral-buffered formalin at ambient temperature for 24 h and washed with PBS. Thereafter, the fixed samples were rehydrated in graded ethanol (EtOH) solutions (3 min each in 100% 2×; 95% 1×; 70% 1×; and 50% 1×) and embedded in paraffin. Paraffin-embedded tissues were sectioned into 8-μm-thick sections, which were then mounted onto poly-l -lysine-coated slides. The slides were boiled in 10 mM sodium citrate for 10 min and chilled on ice for 20 min. Thereafter, they were washed with 3% hydrogen peroxide for 10 min and blocked for 1 h at ambient temperature. The slides were incubated with the primary antibody and then with an anti-rabbit IgG antibody (secondary antibody). Finally, the slides were immunostained using the ABC detection kit in accordance with the manufacturer’s instructions and stained with DAB. The slides were examined under a Nikon Eclipse TE-2000-E confocal microscope (Tokyo, Japan).
7
Phage Immunostaining in Breast Cancer Cell Lines
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Example 3
MCF-7, MCF-7/TGFβ, MDA-MB-231 and Hs578T (breast fibroblasts) cells were seeded in a 4-well chamber overnight. On the next day, cells were fed with fresh medium. LGLRGSL (SEQ ID NO: 1) (E11) (108 pfu) was added in fresh medium and incubated at RT for one hour. After removing the unbound phages, cells were washed with wash buffer (0.1% tween-20 in PBS) three times and fixed with 4% formaldehyde for 15 minutes at 37° C. Cells were permeabilized with 0.2% Triton X-100 at RT for 10 minutes. Reagent was removed and cells were washed with TBS 3 times. Before incubation with anti-phage antibody, cells were treated with blocking buffer for 30 minutes at RT. Cells were incubated with M13-pIII monoclonal antibody for 1 hour at RT, washed and incubated with the secondary goat anti-mouse IgG antibody labeled with Alexa Flour® 488 (Molecular Probes) at a dilution of 1:500 in PBS containing 1% BSA for 45 minutes at RT. Cells were washed three times after secondary antibody treatment. TOTO-3 was used for nucleus staining. Cells were covered with cover slides with Prolong Gold Anti-fade Reagents. Nail polish was used to seal the slide. Pictures were taken by using the NIKON eclipse TE 2000-E confocal microscope.
8
3D Matrigel Culture: Confocal Imaging
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3D culture experiments were performed by seeding cells onto matrigel (BD Biosciences) supplemented with 1.6 μg/ml collagen I (BD BioScience). Cultures were grown for 6–8 days in medium containing 2% matrigel, 2% FBS and 5 ng/ml EGF32 (link), 65 (link). Cells were then fixed with 4% formaldehyde and stained directly in situ with rhodamine phalloidin (Life Technologies) and DAPI. Slides were then visualized using a Nikon Eclipse TE-2000E confocal microscope equipped with the EZ-C1 3.90 software.
9
Macrophage Morphology Changes after GBS Infection
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Cells were seeded in Ibidi 2-well μ-slide (Ibidi GmbH), differentiated into macrophages and GBS isolates (isolates 203, 211, 231) were added for 3 h as described in 2.3 and 2.4. Non-infected macrophages were used as a control. Following infection, cells were washed and fresh RPMI 1640 supplemented with 2% FBS, 100 μg/ml gentamicin and 5 μg/ml penicillin G was added to kill extracellular bacteria. Cells were then incubated at 37°C and 5% CO2 for an additional 3 days to achieve maximal morphological changes. After incubation, cells were washed with RPMI and CellMask Green Plasma Membrane Stain (Invitrogen) was added at a dilution of 1:500 for 15 min to stain the membranes. Cells were visualized using a Nikon Eclipse TE2000-E confocal microscope with 60x objective. In addition, morphological changes were quantified in a blinded fashion and number of cells with altered morphology was enumerated.
10
Phage Binding Assay in Breast Cell Lines
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MCF-7, MCF-7/TGFβ, MDA-MB-231 and Hs578T (breast fibroblasts) cells were seeded in 4-well chamber overnight. On next day, cells were fed with fresh medium. Phage LGLRGSL (E11) (108 pfu) was added in fresh medium and incubated at RT for 1 h. After removing the unbound phages, cells were washed with wash buffer (0.1% tween-20 in PBS) three times and fixed with 4% formaldehyde for 15 min at 37°C. Thereafter, cells were permeabilized with 0.2% Triton X-100 at RT for 10 min. Then, cells were washed with TBS 3 times. Before incubation with anti-phage antibody, cells were treated with blocking buffer for 30 min at RT. Cells were incubated with M13-pIII monoclonal antibody for 1 h at RT, washed and incubated with the secondary goat anti-mouse IgG antibody labeled with Alexa Flour® 488 (Molecular Probes) (1:500 in PBS containing 1% BSA) for 45 min at RT. Subsequently, cells were washed three times and stained with TOTO-3 for nucleus staining. Prolong Gold Anti-fade Reagents was used on the cells which were then covered with cover slides. Pictures were taken by using the NIKON eclipse TE 2000-E confocal microscope. The fluorescence intensity of the images was quantified using image J software.
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