Time-lapse ion imaging assay for the chemical-based fluorescent probes, ie, DAF to measure cytosolic NO, FluoZin-3 to measure cytosolic Zn 2+ , and RhodZin-3 to measure mitochondrial Zn 2+ , was carried out as previously described 24-26 with slight modifications. The cells were stained with specific indicators, eg, DAF (10 μM, 40 minutes at 37°C), FluoZin-3 (5 μM, 40 minutes at room temperature), or RhodZin-3 (5 μM, 30 minutes at 4°C), in a Ca 2+ -free loading buffer and mounted in a recording chamber of an inverted microscope (IX-71, Olympus) with ×40 oil objectives (NA = 1.35, U/340) or a confocal microscope (LSM 5 Pascal, Zeiss, Germany) with ×63 (NA = 1.4) oil objectives. The cells stained with DAF, FluoZin-3, or RhodZin-3 were illuminated by a Xe lamp within a monochrometer (Polychrome V, T.I.L.L. Photonics, Germany) at 495/480 nm (for DAF/FluoZin-3) or at 550 nm (for RhodZin-3) or 543 nm laser under confocal imaging. The emission images of FluoZin-3/DAF or RhodZin-3 were obtained through the 505-520 nm band pass or 560 nm long pass filters, respectively. The images were collected using a high-speed cooled CCD camera (Orca AG, Hamamatsu Photonics, Japan) and recorded using SimplePCI 6.0 (Compix Inst.) or by a confocal microscope imaging system (LSM 5 Pascal, Zeiss, Germany). All live-cell imaging experiments were repeated thrice on different coverslips.
Lsm 5 pascal
Manufactured by Zeiss
Sourced in Germany, United States
The LSM 5 Pascal is a laser scanning microscope. It is designed for high-resolution imaging and analysis of samples.
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Lab products found in correlation
Lsm 510 meta, by Zeiss (8 mentions)
Vectashield, by Vector Laboratories (7 mentions)
Axiovert 200m, by Zeiss (6 mentions)
Lsm 710, by Zeiss (6 mentions)
Triton x 100, by Merck Group (5 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (5 mentions)
Aim software, by Zeiss (4 mentions)
Lsm image browser software, by Zeiss (4 mentions)
Fluoromount g, by Southern Biotech (4 mentions)
Propidium iodide, by Merck Group (4 mentions)
Hoechst 33342, by Merck Group (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Lsm 880, by Zeiss (4 mentions)
Axiovert 200m microscope, by Zeiss (3 mentions)
Oct compound, by Sakura Finetek (3 mentions)
Apoptag red in situ apoptosis detection kit, by Merck Group (3 mentions)
Fluo 4 am, by Thermo Fisher Scientific (3 mentions)
Laser scanning microscopy image examiner software, by Zeiss (2 mentions)
C apochromat 40 1.2 w objective, by Zeiss (2 mentions)
Plan neofluar 10 0, by Zeiss (2 mentions)
267 protocols using lsm 5 pascal
1
Fluorescent Probes for Live-Cell Imaging
2
Immunofluorescence Analysis of Tight and Adherens Junctions
3
Immunohistochemical Analysis of Rat Hearts
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Five hearts from STZ and 5 hearts from control rats were removed and flash frozen with liquid N2 and stored at −80°C until the tissue was processed. The frozen hearts were serially cryosectioned at 18 μm from the posterior to anterior sides in the coronal plane to obtain the four-chamber view. Immunohistochemistry was used to label different regions of the heart using primary antibodies (Supplementary Table S1 ) as previously described (Yanni et al., 2010 (link)). Frozen sections were fixed in 10% neutral buffered formalin, washed in PBS (NaCl, 8 g; KCl, 0.2 g; NaHPO4, 1.44 g; KHPO4, 0.24 g; deionised water, 1 l), permeabilized in 0.1% Triton X-100 in PBS, washed in PBS, blocked with 3% BSA in PBS and incubated in primary antibodies at 4°C overnight. The following day, after PBS wash, secondary antibodies and TUNEL assay (to some sections) were applied (Supplementary Table S3 ) for 120 min and washed in PBS. Tissue sections were then mounted with an anti-fade medium for fluorescence (Vectashied; Vector Lab, Peterborough, United Kingdom), covered with coverslips and sealed with nail varnish. Confocal images were acquired using a laser-scanning microscope (Zeiss LSM 5 PASCAL). Images were acquired using the following conditions: 488 nm excitation and 505–530 nm emission for FITC and TUNEL, and 543 nm excitation and >560 nm emission for Cy3.
4
Quantifying Dendritic Complexity via Sholl Analysis
5
Morphological Characterization of Drug-Loaded Fiber Mats
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Morphologies of drug-loaded fiber mats were observed by an optical microscope (BH-2, Olympus, Center Valley, PA, USA); a field-emission scanning electron microscope (FE-SEM) (Nova NanoSEM 450, FEI, Hillsboro, OR, USA) with the prior sputter coating of 3.5 nm thick Au (Hummer VI, Anatech, Hayward, CA, USA); and a transmission electron microscope (TEM, CM-100, Philips, Eindhoven, Netherlands). For cross-sectional TEM images of fibers, sections of fibers were prepared by embedding fibers in Spurr's epoxy resin (Ultra Bed Low Viscosity Epoxy Kit, Electron Microscopy Sciences, Hatfield, PA, USA) and sectioning in about 90 nm thickness using an ultramicrotome (Ultracut UCT, Leica, Germany). Fluorescence images of Dox-loaded webs were observed with a laser scanning confocal microscope (LSM 5 Pascal, Zeiss, Oberkochen, Germany) and Olympus IX73 inverted fluorescent microscope system. Images were analyzed by ImageJ software (version 1.46r, NIH, USA).
6
Immunocytochemical Quantification of Cellular Signaling
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Immunocytochemistry was performed on samples obtained by peritoneal lavage cytocentrifugation followed by adherence of peritoneal macrophages to tissue culture dishes. The slides were then fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich), and stained with anti-macrophage-specific marker (Mac3; BD Pharmingen, San Jose, CA), mouse monoclonal anti-phospho-STAT6, anti-STAT6, or anti-PPARγ antibody overnight at 4 °C. Subsequently, cells were washed with phosphate-buffered saline three times and incubated with fluorescent isothiocyanate-conjugated donkey anti-rabbit IgG (Jackson ImmunoReseach, West Grove, PA, USA). The slides were mounted in Vectashield mounting medium with DAPI (Vector Laboratories, Inc., Youngstown, OH, USA). All slides were imaged using a confocal microscope (LSM5 PASCAL; Carl Zeiss, Jena, Germany) equipped with a filter set with excitation at 488 and 543 nm. The phosphor-STAT6, STAT6, and PPARγ stainings were quantified by creating masks and measuring the mean fluorescence intensity of each staining using the laser scanning microscopy image examiner software (Carl Zeiss).
7
Comprehensive Microscopic Analysis of Tissue Perfusion
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To assess vessel perfusion, the hypoxic areas, apoptosis, and pericyte coverage, cryosections were processed and were visualized and imaged using an all-in-one microscope system (BZ8000; Keyence Corporation, Osaka, Japan) with a 20× objective lens (Plan Fluor 20×: numerical aperture [NA] = 0.75; working distance = 1.0 mm; Nikon Corporation, Tokyo, Japan).
The infarct area that was stained with rhodamine-conjugated G. simplicifolia lectin I was placed on a microscope slide with Vectashield® and it was flattened with a cover slip. New vessels in the infarct area were imaged using confocal microscopy (LSM 5 Pascal; Carl Zeiss AG, Oberkochen, Germany) by scanning 12 layers (with 2.5 μm between the adjacent layers) using a 20 × objective lens (EC Plan-Neofluar 20 × : NA = 0.5; working distance = 2.0 mm; Carl Zeiss AG).
The infarct area that was stained with rhodamine-conjugated G. simplicifolia lectin I was placed on a microscope slide with Vectashield® and it was flattened with a cover slip. New vessels in the infarct area were imaged using confocal microscopy (LSM 5 Pascal; Carl Zeiss AG, Oberkochen, Germany) by scanning 12 layers (with 2.5 μm between the adjacent layers) using a 20 × objective lens (EC Plan-Neofluar 20 × : NA = 0.5; working distance = 2.0 mm; Carl Zeiss AG).
8
Neuron Morphology Quantification
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XY scans (×20 objective, 1,024 × 1,024 pixels) were taken on a confocal laser‐scanning microscope (LSM 5 Pascal; Zeiss, Germany). A grid of concentric circles spaced 15 μm apart was placed around the cell soma of GFP‐expressing neurons, and the number of dendrites crossing each circle was counted manually on images thresholded in ImageJ. The average number of intersections was calculated for approximately 10 cells per each condition, in each of three or four independent experiments. All experiments were performed in a blinded manner.
9
3D Confocal Imaging and Analysis
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3D image z-stacks were collected on an inverted laser scanning confocal microscope (LSM5 Pascal, Carl Zeiss, Thornwood, NY, USA) using either a Plan-Neofluar 10×/0.3, Plan-Neofluar 40×/0.75 or C-Apochromat 40×/1.2 W objective (Carl Zeiss, Thornwood, NY, USA). The EGFP reporter was excited with the 488 nm laser line using the FITC filter and all other imaging parameters were as described in Kasemeier-Kulesa et al. (2005) (link). Images were collected, processed and analyzed using AIM software (Carl Zeiss, Thornwood, NY, USA). Statistical analysis was performed using the Student's t-test.
10
Visualization of β-catenin Translocation
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Immunocytochemistry was carried out as previously described.6 , 23 To evaluate the effect of PTEN4A or PTEN mutants on the TGFβ‐induced translocation of β‐catenin, H358ON cells expressing Dox‐dependent PTEN mutants were incubated with anti‐β‐catenin antibody, followed by SAv‐594‐conjugated anti‐mouse antibody. Nuclear staining was carried out using Hoechst 33342. The distribution of β‐catenin was determined by confocal laser scanning microscopy (LSM 5 PASCAL; Carl Zeiss, Jena, Germany). The fluorescence intensities of β‐catenin and the nucleus were evaluated by using imaging software (LSM Software ZEN 2008; Carl Zeiss), which plotted fluorescence intensities over a random cross‐section of the cells.24 , 25 The subcellular distribution of PTEN was determined by confocal laser scanning microscopy. A minimum of five randomly selected high‐power fields were examined per sample to measure fluorescence intensity in the nucleus and the cytoplasm.26 To determine localization of β‐catenin in the cell membrane, double immunostaining was carried out for β‐catenin and E‐cadherin in the cells.27
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