Immunofluorescence experiments were performed as previously described43 (link) using permanox Labtek chamber slides as support. Briefly, cells were fixed with 4% paraformaldehyde for 10 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS (Sigma-Aldrich, St. Louis, MO). Cells were then blocked with 5% BSA in PBS for 30 min at room temperature and incubated overnight with the following primary antibodies: anti-Gli1 H300 (sc-20687, Santa Cruz Biotechnology Inc.), anti-Gli1 (AF3455, R&D Systems), anti- ABCB1 (MDR1, D-11; sc-55510 Santa Cruz Biotechnology Inc.) and anti‐ABCG2 H-70 (sc‐2582; Santa Cruz Biotechnology, Inc.) diluted in blocking solution. Secondary antibodies conjugated with Alexa Fluor 488 or 594 were purchased from Molecular Probes (Invitrogen) and diluted 1:400 and 1:200, respectively, in blocking solution. Nuclei were Hoechst-counterstained and cover slips were mounted with fluorescence mounting medium (Prolong Gold, Thermo Fisher Scientific, MA, USA). Images were acquired using a FV1200 MPE laser scanning confocal microscope (Olympus) with a UPlanSAPO 20x/0.75 NA objective. Imaris 8.1 software (Oxford Instruments, https://imaris.oxinst.com/ ) was used for image-processing.
Imaris 8
Manufactured by Oxford Instruments
Sourced in Switzerland, United Kingdom
Imaris 8 is a comprehensive software suite for 3D and 4D image visualization, analysis, and processing of large datasets from a variety of microscopy techniques. The software provides advanced tools for segmentation, tracking, and quantification of biological structures.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (4 mentions)
Lsm 880 airy nlo, by Zeiss (3 mentions)
Autoquant x3, by Oxford Instruments (2 mentions)
Chameleon vision 2, by Coherent Inc (2 mentions)
A1 confocal microscope, by Nikon (2 mentions)
Eclipse ti inverted microscope, by Nikon (2 mentions)
Fv500, by Olympus (2 mentions)
Cy3 streptavidin, by Jackson ImmunoResearch (2 mentions)
Cd45 biotin 30 f11, by BioLegend (2 mentions)
Cd11c alexa fluor 647 n418, by BioLegend (2 mentions)
Nk1.1 biotin pk136, by BioLegend (2 mentions)
Slowfade diamond antifade, by Thermo Fisher Scientific (2 mentions)
Tcs sp8, by Leica (2 mentions)
Cytofix cytoperm buffer, by BD (2 mentions)
Lsm 800, by Zeiss (2 mentions)
Anti gli1 h300, by Santa Cruz Biotechnology (1 mentions)
Anti abcg2 h 70, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 or 594, by Thermo Fisher Scientific (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
35 protocols using imaris 8
1
Immunofluorescence Staining of Gli1 and ABC Transporters
2
Proximity Ligation Assay for Immune Cell Signaling
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Immunofluorescence analysis of RBL-2H3 cells and BMMC was performed as described (80) . PLA was performed using a Duolink in situ kit (Sigma-Aldrich). Cultured BMMCs (100,000 cells) were plated on coverslips coated with fibronectin (10 g/ml; Sigma-Aldrich) for 45 min in MnCl 2 (1 mM)-containing medium and maintained at 37°C in a humidified atmosphere. After PMA/ionomycin (20 nM:1 M) stimulation for indicated time points, cells were fixed, Downloaded from https://www.science.org on September 06, 2024 quenched, permeabilized (16) , and blocked for 1 hour at 37°C before incubation with primary Abs for 2 hours at room temperature. PLA was then performed according to the manufacturer's instructions. Complexes were analyzed using an LSM 780 Zeiss confocal microscope (Carl Zeiss) at 63× steps of a 0.38-m microscope. Images were processed with Imaris 8.1 software (Bitplane, Oxford Instruments), and the spots per nucleus were quantified. To evaluate membrane or cytoplasmic localization, cells were additionally labeled with Alexa Fluor 488-wheat germ agglutinin (Thermo Fisher Scientific) before the permeabilization procedure, and dots were evaluated for membrane-proximal or cytoplasmic localization using Imaris 8.1 software counting at least 30 cellular sections.
3
Curvature-Dependent Septin and AH Adsorption
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Experiments measuring the adsorption of septins and AH domains on different curvatures were performed as previously published (Cannon et al., 2019) . Reactions (with a final buffer composition of 100 mM KCl, 50 mM Hepes pH 7.4, 1 mM BME, 0.1% methyl-cellulose, and 0.01% BSA) were incubated in a plastic chamber (Bridges and Gladfelter, 2016) glued onto a polyethylene glycol (PEG)-coated passivated coverslip for one hour to reach equilibria. Confocal images of fluorescent-tagged protein adsorbed onto curved supported bilayers on microspheres were acquired using a spinning disc (Yokogawa W1) confocal microscope (Nikon Ti-82 stage) using a 100x Plan Apo 1.49 NA oil lens and a Prime 95B CMOS camera (Photometrics). Raw images were analyzed using Imaris 8.1.2 software (Bitplane AG) as previously described (Cannon et al., 2019) . Boxplots were generated using R version 3.2.2 (R Foundation for Statistical Computing; R studio 0.99.467) with ggplot2 package (Wickham et al., 2007 (Wickham et al., , 2015)) .
4
Imaging Septin Dynamics on Curved Membranes
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Images of fluorescent-tagged septins adsorbed onto curved supported bilayers on microspheres were acquired using a spinning disc (Yokogawa W1) confocal microscope (Nikon Ti-82 stage) using a 100× Plan Apo 1.49 NA oil lens and a Prime 95B CMOS camera (Photometrics). Images were analyzed using Imaris 8.1.2 software (Bitplane AG) as previously described (Bridges et al., 2016 (link); Cannon et al., 2019 (link)).
5
Image Deconvolution for Microscopy
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After acquisition, 3D stacks were processed for deconvolution. This image processing technique was performed to restore the imaged objects usually degraded by blurring and noise, artifacts inherent to any image acquisition system. The degree of blurring of a single, sub-resolution point-like object was considered as a measure of the optical system's quality and the blurred 3D image of this single point light source was defined by the Point Spread Function (PSF) [11] (link). Therefore, to counteract the effects of this blurring and to optimize image quality and subsequent quantifications, all images were processed using image processing software package (Huygens Professional, Scientific Volume Imaging b.v., Hilversum, The Netherlands) or image deconvolution software (Auto-Quant X3, Bitplane AG, Zurich, Switzerland).
Once images were processed to maximize quality, the relevant parameters were measured using image visualization and analysis software (Imaris 8.3.4, Bitplane AG, Zurich, Switzerland; Fiji ImageJ, Bethesda, Maryland, USA; IllucidaFX, Los Angeles, USA). Full descriptions of methodology and parameters studied are given in the subsequent sections.
Once images were processed to maximize quality, the relevant parameters were measured using image visualization and analysis software (Imaris 8.3.4, Bitplane AG, Zurich, Switzerland; Fiji ImageJ, Bethesda, Maryland, USA; IllucidaFX, Los Angeles, USA). Full descriptions of methodology and parameters studied are given in the subsequent sections.
6
Deconvolution and Image Analysis for Enhanced Microscopy
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After acquisition, 3D stacks were processed for deconvolution. This image processing technique was performed to restore the imaged objects usually degraded by blurring and noise, artifacts inherent to any image acquisition system. The degree of blurring of a single, sub-resolution point-like object was considered as a measure of the optical system’s quality and the blurred 3D image of this single point light source was defined by the Point Spread Function (PSF) [11 (link)]. Therefore, to counteract the effects of this blurring and to optimize image quality and subsequent quantifications, all images were processed using image processing software package (Huygens Professional, Scientific Volume Imaging b.v., Hilversum, The Netherlands) or image deconvolution software (AutoQuant X3, Bitplane AG, Zurich, Switzerland).
Once images were processed to maximize quality, the relevant parameters were measured using image visualization and analysis software (Imaris 8.3.4, Bitplane AG, Zurich, Switzerland; Fiji ImageJ, Bethesda, Maryland, USA; IllucidaFX, Los Angeles, USA). Full descriptions of methodology and parameters studied are given in the subsequent sections.
Once images were processed to maximize quality, the relevant parameters were measured using image visualization and analysis software (Imaris 8.3.4, Bitplane AG, Zurich, Switzerland; Fiji ImageJ, Bethesda, Maryland, USA; IllucidaFX, Los Angeles, USA). Full descriptions of methodology and parameters studied are given in the subsequent sections.
7
Second-Harmonic Generation Imaging of Collagen Fibers
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Second-harmonic generation (SHG) imaging was carried out using a high-speed multiphoton confocal microscope (A1R + MP, Nikon, Tokyo, Japan) with a laser oscillator (wavelength 690–1040 nm, repetition rate 80 MHz, and pulse width 70 fs) (Mai Tai eHP, Spectra-Physics, Andover, MA) and a water-immersion objective lens (numerical aperture 1.1) (CFI75 Apo 25 × W MP, Nikon). The excitation wavelength used for collagen fiber observations was 880 nm. Image acquisition, orthogonal view processing, and trimming were carried out using NIS-Elements ver. 4.0 (Nikon). As shown in the legends to the corresponding figures, the look-up tables for this software were used to adjust the brightness and contrast of a number of images on the basis of the shared parameters of associated images. From the image obtained, a square region of 200 µm × 200 µm at the implant neck (a, d), a region at the central part (b, e) and a region at the implant apex (c) were extracted as the regions of interest (Fig. 1 B). High-precision image analysis software (Imaris8.4, Bitplane AG, Zürich, Switzerland) was used to trace and measure the angles of the collagen fiber bundles.
8
Quantifying Collagen Fiber Bundles in Calcified Tissues
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Second harmonic generation (SHG) images were obtained using a multiphoton confocal microscopy system (LSM 880 Airy NLO; Carl Zeiss, Oberkochen, Germany) with an excitation laser (Chameleon Vision II, wavelengths: 680-1080 nm; repetition rate: 80 MHz; pulse width: 140 fs; Coherent Inc. Apochromat 10×/0.8 M27; Carl Zeiss, Oberkochen, Germany). The excitation wavelength for observation of collagen fibers was 880 nm. Software (ZEN, Carl Zeiss, Oberkochen, Germany) was used for image acquisition. After image acquisition, the collagen fiber bundles in the calcified area of interest were traced using Imaris 8.4 (Bitplane AG, Switzerland), and the fiber bundle diameters were measured.
9
Collagen Fiber Orientation Analysis via SHG Imaging
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Second harmonic generation (SHG) images were obtained using a multiphoton confocal microscopy system (LSM 880 Airy NLO, Carl Zeiss, Oberkochen, Germany) with excitation laser (Chameleon Vision II, wavelengths: 680–1080 nm; repetition rate: 80 MHz pulse width: 140 fs, Coherent Inc., Santa Clara, CA, USA) and objective lens (Plan-Apochromat 10x/0.8 M27, Carl Zeiss, Oberkochen, Germany). The excitation wavelength for observing collagen fibers was 880 nm. From the image obtained, a region of 200 μm × 200 μm square at the implant neck (A and F) was extracted as the region of interest. High-precision image analysis software (Imaris8.4, Bitplane AG, Zürich, Switzerland) was used to trace and measure the angles of the collagen fiber bundles.
10
Biofilm Live/Dead Visualization and Quantification
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In situ grown biofilms were fluorescent labeled using the LIVE/DEAD TM BacLight™ Bacterial Viability Kit (Thermo Fisher Scientific, Braunschweig, Germany). The fluorescent dyes Syto9 ® and propidium iodide were simultaneously applied as 1:4000 dilution in PBS according to the manufacturer's recommendations. Specimens were fixed with 2.5% glutaraldehyde (Carl Roth GmbH + Co. KG) and placed in PBS for microscopy. Using the TCS SP8 confocal laser-scanning microscope (CLSM, Leica Microsystems GmbH, Wetzlar, Germany) three-dimensional images with a 400× magnification and a z-step-size of 5 µm were taken at four defined positions per specimen (Fig. 2c). The laser lines 488 nm and 552 nm as well as emission spectra of 500-550 nm and 650-750 nm were used to detect Syto9 and propidium iodide, respectively. The Imaris software package (Imaris 8.4, Bitplane AG, Zurich, Switzerland) was used to quantify biofilm volume and live/dead distribution.
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