Attached cells were fixed by 4% paraformaldehyde for 30 min, and penetrated by Triton X-100 for 15 min, then blocked by BSA for 30 min. DYKDDDK Tag (9A3) mouse mAb (1: 100, Cell signaling) was incubated at room temperature for 1 h. After rinsed by PBS, Anti-mouse IgG1 FITC (1:100, eBioscience) was incubated for 30 min. Slides were mounted by VECTASHIELD Mounting Medium with DAPI (Vector Labs). Images were captured by confocal microscope, with Zeiss LSM Image Examiner (Carl Zeiss).
Lsm image examiner
Manufactured by Zeiss
Sourced in Germany
The LSM Image Examiner is a software tool designed for the analysis and processing of images obtained from Zeiss laser scanning microscopes. It provides users with the ability to view, manipulate, and extract data from microscopy images. The core function of the LSM Image Examiner is to facilitate the examination and evaluation of microscopic samples.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510 meta, by Zeiss (8 mentions)
Lsm 510, by Zeiss (6 mentions)
Lsm 510 meta confocal microscope, by Zeiss (6 mentions)
Lsm 710, by Zeiss (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Fluorescein isothiocyanate labeled goat anti mouse, by Jackson ImmunoResearch (2 mentions)
βiii tubulin, by Promega (2 mentions)
Triton x 100, by Solarbio (2 mentions)
Paraformaldehyde (pfa), by Solarbio (2 mentions)
Anti bip, by Abcam (2 mentions)
Anti giantin, by Abcam (2 mentions)
4 well chamber slide, by Thermo Fisher Scientific (2 mentions)
Anti flag m2, by Abcam (2 mentions)
Prolong antifade reagent, by Thermo Fisher Scientific (2 mentions)
Mouse anti human ve cadherin antibody, by BD (2 mentions)
Lsm 510 meta confocal scanning laser inverted microscope, by Zeiss (2 mentions)
Alexa fluor 594 goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 rabbit anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Eclipse 80i microscope, by Nikon (2 mentions)
38 protocols using lsm image examiner
1
DYKDDDK Tag Immunofluorescence Staining
2
Quantification of ALT-Associated PML Bodies
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The APB detection method used in this study is a modified version of the method used by Yeager et al. (1999) and Henson et al. (2005) and our specific methodology has been reported in earlier studies 8 , 18 , 19 . Briefly, slides were incubated an anti‐PML rabbit polyclonal primary antibody (H‐238, Santa Cruz Biotechnology, Santa Cruz SD) using a 1 in 500 dilution and detected with an Alexa Fluor 488 antibody (Life Technologies, Carlsbad, CA). Telomere DNA was detected using a Cy3‐labelled PNA probe 5′‐CCCTAACCCTAACCCTAA‐3′ (Life Technologies, Carlsbad, CA). The cellular nuclei were stained using DAPI. Cells were imaged using confocal microscopy (Zeiss LSM510; Carl Zeiss, Thornwood, NY). For each colocalized signal, the fluorescent signal from each channel was analysed using the software Zeiss LSM Image Examiner (Version 30115; Carl Zeiss Thornwood, NY). A tumour was designated APB positive when a colocalized focus of telomeric DNA and PML protein within the nucleus was identified in ≥ 5% of the cells. One APB in a cell nucleus qualified the cell as positive. APBs were interpreted as bright yellow colocalized areas with a clear peak signal of at least 100 relative fluorescent intensity (Figure 2 ).
3
Mitochondrial Staining and Confocal Imaging
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Cells were seeded on poly-L-lysine-coated coverslips and maintained in growth medium for 24 h. The cells were stained with 100 nM MitoTracker Red CMXROS (Invitrogen, Carlsbad, CA, USA) for 30 min. The cells were then washed twice with PBS, fixed in 3.7% formaldehyde for 15 min, permeabilised with ice-cold acetone for 5 min, and washed again with PBS. Cell nuclei were then counterstained with DAPI and mounted. The fluorescent signal was observed using a confocal microscope (LSM 510META, Carl Zeiss, Oberkochen, Germany). Zeiss LSM 5 software (v3.2) was used to acquire images and Zeiss LSM Image Examiner (v4,0,0,241) was used for fluorescence image processing.
4
Quantifying Antibiofilm Effects of UMB
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The biofilm was allowed to form on glass and titanium (1 × 1 cm) pieces in the absence and presence of UMB (at 500 μg/ml) as described earlier. Then, the untreated and UMB treated biofilms formed on glass and titanium surfaces were stained with acridine orange (0.1% w/v) at dark condition for 5 min, de-stained, air-dried and imaged at 200X magnification under CLSM (LSM 710, Carl Zeiss, Germany). Further, Zeiss LSM Image Examiner and Zen 2009 image software (Carl Zeiss, Germany) were used for image processing and z-stack analysis, respectively. COMSTAT software (gifted by Dr. Claus Stenberg, Technical University of Denmark) was also employed to quantify biofilm entities such as biomass, maximum thickness, and surface to volume ratio for understanding the extent of antibiofilm effect of UMB over different surfaces.
5
Immunohistochemical Analysis of Neuromuscular Junctions
6
Immunofluorescence Assay for Epithelial-Mesenchymal Transition
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KYSE30 cells were treated with si‐MTA2 for 24 h, fixed with 4% paraformaldehyde (Solarbio) for 30 min, permeabilized with 0.1% Triton X‐100 (Solarbio) for 15 min, and blocked with 5% BSA for 30 min. The cells were then incubated with antibodies, including MTA2, E‐cadherin, N‐cadherin, and vimentin (CST) for 2 h at room temperature, followed by further incubation at room temperature for 1 h with rabbit IgG (Alexa Fluor 546, green). Nuclear DNA was labeled in blue with DAPI (Beyotime). Images were captured by confocal microscopy on a Zeiss LSM Image Examiner (Carl Zeiss).
7
Characterizing Surface Roughness and Wettability
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Surface roughness was estimated using laser scanning confocal microscope (Zeiss LSM 5 Pascal, Carl Zeiss Inc., Oberkochen, Germany) with imaging software (Zeiss LSM Image Examiner, Carl Zeiss Inc.). The multi-argon laser emits light with a wavelength of 633 nm. This allows the calculation of the arithmetic mean of surface roughness from a mean plane in the sampling area (900×900×350 µm). To evaluate surface wettability, water contact angle was measured by sessile drop method at room temperature. A video camera with an image analyzer (Phoenix 300, Surface Electro Optics, Seoul, Korea) visualized the shape of the drop and provided the contact angle. Three probe liquids of different polarities were used: 1-bromonaphthalene, formamide, and deionized water. The volume of liquid drops was controlled using an instrument with a computerized interface and pictures of the drops were taken as soon as they landed on the surfaces using a video camera. Right and left contact angles of each drop were automatically averaged to give one contact angle per drop. Then, the surface free energy was calculated using three contact angle values from three different probe liquids according to the van Oss model. Each experiment was repeated 5 times for 3 specimens of each experimental group. The surface energy was calculated using the Owens-Wendt equation [6 ].
8
Histological Analysis of Neuromuscular Junctions
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Extensor digitorum longus (EDL) muscles were dissected, pinned in mild stretch, and fixed by immersion for 20 minutes in 4% paraformaldehyde with PBS (pH 7.4). After rinsing in PBS, muscles were soaked in 15% sucrose with PBS (overnight at 4°C), followed by 30% sucrose with PBS for at least 24 hours. Muscle tissues were embedded in OCT and sectioned with a cryostat (HM 550; Microm GmbH) into 40-mm sections and placed on glass slides for staining. For a presynaptic marker, sections were incubated with βIII-tubulin (1:500; Promega, catalog G7121) antibody overnight at 4°C, followed by secondary fluorescein isothiocyanate–labeled goat anti-mouse (1:100; Jackson ImmunoResearch, catalog 115-095-205) antibody. Rhodamine bungarotoxin (1:40, Thermo Fisher Scientific,, catalog B13423) was used as a postsynaptic marker. Stained sections were examined under fluorescence and confocal microscopy. The Zeiss LSM Image Examiner version 1.0.0.241 (Carl Zeiss) was used for imaging analysis. We quantitated pre- over postsynaptic NMJ areas, and this method has shown to be effective in detecting differences in NMJ connectivity in published studies (64 (link)).
9
Visualizing miPEP858a Uptake in Arabidopsis Roots
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For miPEP858a uptake assays in Arabidopsis roots, fluorescent carboxyfluorescein (5-FAM)-labeled miPEP858a at the N-terminus was purchased from Link Biotech (95%-98% purity). Four-day-old Arabidopsis WT, chc mutants' seedlings were incubated with 5-FAM-miPEP858a (50 μM) in MG buffer (10-mM MgCl 2 buffer, pH 5.8) at 22°C for 12 h. After treatment, the seedlings were washed 3 times by gentle shaking for 5 min in MG buffer, and the roots were analyzed using a confocal microscope (Zeiss LSM710, Zeiss LSM Image Examiner Version 4.2.0.121, CarlZeiss) at an excitation 495 nm/emission 545 nm and solid-state laser (10% of 10 mW) for both FAM and PI. Root meristematic, elongation zone was observed under Leica microscope (LAS version 4.12.0, Leica Microsystems).
10
Immunofluorescence Assay of EMT Markers
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KYSE30 cells were treated with si-MTA2 for 24 h, fixed with 4% paraformaldehyde (Solarbio, China) for 30 min, permeabilized with 0.1% Triton X-100(Solarbio, China) for 15 min, and blocked with 5% BSA for 30 min. Then, the cells were incubated with antibodies, including MTA2,E-cadherin N-cadherin and Vimentin (CST, USA) for 2 h at room temperature, followed by further incubation at room temperature for 1 h with rabbit IgG (Alexa Fluor 546, green, USA). Nuclear DNA was labeled in blue with DAPI (Beyotime, China). Images were captured by confocal microscopy, with a Zeiss LSM Image Examiner (Carl Zeiss, Germany).
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