After the last time point of in vivo U‐LSOM imaging, mice were euthanized and their entire skull bone extracted and sliced as described.(27 ) Consecutive slices of 150 μm thickness were generated throughout the entire bone. For confocal imaging, slices covering or proximal to the coronal suture were selected. For immunostaining, individual slices were first incubated in blocking solution (0.2% Triton X‐100, 10% donkey serum in PBS) overnight at 4°C and then stained with primary antibodies (Endomucin, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA; Sc‐65495) in blocking solution for 3 days at 4°C. Tissues were then washed in 0.2% Triton‐X‐100/PBS and stained with secondary antibodies (Alexa Fluor 488 donkey anti‐rat immunoglobulin G [IgG] Antibody, 1:400; Thermo Fisher Scientific, Waltham, MA, USA; A‐21208) for another 3 days at 4°C in blocking solution. Immunostained slices were washed in 0.2% Triton‐X‐100/PBS overnight and incubated in RapiClear 1.52 for 12 to 16 hours. Confocal microscopy was performed with a 10× objective (HCX PL FLUOTAR; Leica, Wetzlar, Germany) on a Leica SP8 Leica confocal microscope. Image stacks (x: 3070–3080 μm, y: 9300–10300 μm, z: 125–145 μm) were acquired with a pixel size of 2.27 μm and a Z‐step size of 3 μm. Imaris software (Oxford Instruments plc, Abingdon, UK) was used to render confocal image stacks into 3D reconstructions (Fig. 3B ).
Hcx pl fluotar
Manufactured by Leica
Sourced in Germany
The HCX PL FLUOTAR is a series of high-quality microscope objectives manufactured by Leica. These objectives are designed to provide excellent optical performance for a variety of microscopy applications. The core function of the HCX PL FLUOTAR objectives is to facilitate high-resolution imaging and analysis of samples.
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Lab products found in correlation
Imaris software, by Oxford Instruments (1 mentions)
Endomucin, by Santa Cruz Biotechnology (1 mentions)
Dmi6000 microscope, by Leica (1 mentions)
Tcs spe confocal microscope, by Leica (1 mentions)
Hcx apo l, by Leica (1 mentions)
Dmlm microscope, by Leica (1 mentions)
Invia raman system, by Renishaw (1 mentions)
Dmire2 microscope, by Leica (1 mentions)
Lab tek chamber, by Thermo Fisher Scientific (1 mentions)
L5 filter cube, by Leica (1 mentions)
Orca 05g, by Hamamatsu Photonics (1 mentions)
Clean microscope coverslips, by Marienfeld (1 mentions)
Quanta 650, by Thermo Fisher Scientific (1 mentions)
Nexion 2000 p, by PerkinElmer (1 mentions)
Ft ir 6700 spectrophotometer, by Jasco (1 mentions)
Dm6000b microscope, by Leica (1 mentions)
Application suite x software, by Leica (1 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (1 mentions)
Application suite x, by Leica (1 mentions)
Tcs sp5 confocal system, by Leica (1 mentions)
13 protocols using hcx pl fluotar
1
Confocal Imaging of Skull Bone Slices
2
Visualizing β-catenin-GFP Dynamics
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Cells were viewed with a 20×/0.4NA dry objective (HCX PL Fluotar) on a wide-field DMI6000 microscope (Leica) to assess β-catenin-GFP localization and cell migration in HGF and wound healing experiments.
3
Zebrafish Embryo Lipoplex Delivery
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Zebrafish (Danio rerio, strain AB/TL) were maintained and handled according to the guidelines from the Zebrafish Model Organism Database (http://zfin.org ) and in compliance with the directives of the local animal welfare committee of Leiden University. Fertilization was performed by natural spawning at the beginning of the light period, and eggs were raised at 28.5°C in egg water (60 µg mL−1 Instant Ocean sea salts). Mingle‐ or single‐lipoplex formulations (0.2 ng mRNA in 2 nL for each embryo) were injected into the hindbrain ventricle of 48 h post fertilization (hpf) zebrafish embryos. Embryos were anesthetized in 0.01% tricaine and embedded in 0.4% agarose containing tricaine before injection. Then, embryos were removed from the agarose. At indicated time‐points after injection, embryos were embedded again and imaged using confocal microscopy. Confocal z‐stacks were captured on a Leica TCS SPE confocal microscope, using a 10 × air objective (HCX PL FLUOTAR) or a 40 × water‐immersion objective (HCX APO L). Laser intensity, gain, and offset settings were identical between stacks and sessions. Images were processed by using the Fiji distribution of Image J.
4
Raman Spectroscopy Characterization Protocol
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Raman spectra were acquired with an inVia Raman system (Renishaw plc) coupled to a Leica DMLM microscope at the University of Edinburgh. The 785 nm (300 mW) excitation laser beam (Toptica) was focused onto the samples using a ×100/0.9 NA objective lens (Leica, HCX PL Fluotar), providing an excitation spot of 1 µm diameter. Raman point spectra were taken at different positions on the samples over the range 100–1200 cm−1 in extended scan mode. The spectra were acquired with 30 s exposure time using a 600 lines /mm−1 diffraction grating. Wire 2.0 software was used for data acquisition.
5
Live-cell Microscopy of Hoechst-EGFP Samples
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Clean microscope coverslips (Marienfeld) or Labtek chambers (Nunc) containing cells were placed in an incubator equilibrated at 37 °C located on a DMIRE2 Leica Microscope controlled by HCImageLive software (Leica). Images from a 100X (N.A. 1.3–0.6) oil-immersion objective (HCX PL FLUOTAR, Leica) were recorded with a Hamamatsu ORCA-05G camera. A4 (Leica) and L5 filter cubes (Leica) were used for excitation and emission of Hoechst 33342 and EGFP fluorescence, respectively.
6
Comprehensive Characterization of FeCO3 Catalyst
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The morphology and elemental composition of the FeCO3 catalyst were determined using a scanning electron microscope equipped with energy-dispersive X-ray spectroscopy (FEI Quanta 650, FEG-SEM). X-ray diffraction patterns were obtained with a PANalytical Empyrean brand powder device at the 2-theta angle between 10–90°. The FT-IR spectrum of FeCO3 was recorded in a Jasco FT-IR-6700 spectrophotometer using the KBr technique within the range of 400–4000 cm−1. Raman spectra were recorded with a Renishaw inVia Qontor using a 532 nm 50 mW diode laser (M2 < 1.1, beam divergence < 0.45 mrad) with 1800 line/mm grating and an x50 long working distance objective lens (Leica HCX PL FLUOTAR, WD = 8 mm, NA = 0.55). The N2 adsorption isotherm was measured at −196 ℃ using a Micromeritics Tristar Orion II 3020 surface area and porosimetry analyzer. Brunauer–Emmet–Teller (BET) method was used to analyze the surface area of the FeCO3 catalyst. The amount of Fe ions leaching into the solution was determined quantitatively by Perkin Elmer NEXION 2000 P brand inductively coupled plasma-mass spectrometer (ICP-MS).
7
Holographic Imaging of Retinal Photoreceptors
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Small sections of retina previously fixed with 4% paraformaldehyde, were teased apart onto a no.1 glass microscope coverslip in PBS resulting in the release of rod outer segments into the solution. A coverslip was placed over these and sealed with nail vanish.
The optical properties of the photoreceptor outer segments were measured using a digital holographic microscope (DHM) (T1000, LyncéeTec, Lausanne, CH) at a laser wavelength of 660 nm and under a 100x oil immersion objective (Leica, HCX PL Fluotar). The retardation provides a proportional measure of the refractive index of the sample with reference to the surrounding medium. A detailed description of the technique can be found in Colomb et al.77 (link). Holographic data were reconstructed into 2-dimensional retardation maps from the samples using the DHM software, Koala (version 4) (LyncéeTec, Lausanne, CH) and phase transects calculated across the outer segments. Phase data and statistical analyses were processed using R (version 2.15.3). Values of retardation per micron were recorded from quantitative phase measurements of 10 outer segments from each animal along with diameters of the each cell.
The optical properties of the photoreceptor outer segments were measured using a digital holographic microscope (DHM) (T1000, LyncéeTec, Lausanne, CH) at a laser wavelength of 660 nm and under a 100x oil immersion objective (Leica, HCX PL Fluotar). The retardation provides a proportional measure of the refractive index of the sample with reference to the surrounding medium. A detailed description of the technique can be found in Colomb et al.77 (link). Holographic data were reconstructed into 2-dimensional retardation maps from the samples using the DHM software, Koala (version 4) (LyncéeTec, Lausanne, CH) and phase transects calculated across the outer segments. Phase data and statistical analyses were processed using R (version 2.15.3). Values of retardation per micron were recorded from quantitative phase measurements of 10 outer segments from each animal along with diameters of the each cell.
8
Microscopic Leaf Analysis Protocol
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Whole leaves were placed in a Petri dish, illuminated by two lateral light sources. Images were obtained under bright field with a Magnifier MZ16F stereomicroscope in conjunction with las , version 4.12 (Leica, Wetzlar, Germany). Sections of 1 cm2 were cut from whole leaves, placed between a glass slide and a coverslip and observed under a Leica 500 microscope using las , version 4.9 and a 40× objective (40×/0.75 HCX PL Fluotar; Leica). Images were obtained under bright field and under an UV‐A filter (340–380 nm excitation; 425 nm emission).
9
Microscopic and Flow Cytometric Analysis of E. coli
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For microscopy assay, 5 µL E. coli cells were mounted directly on microscope slides and observed immediately under microscope. Microscopy was carried out by using a 100x
Leica HCX PL FLUOTAR oil objective lens on a Leica DM6000 B microscope. Images were acquired by Leica Application Suite X software. Image analysis was carried out by ImagJ software. Flow cytometry analysis was carried out with Attune TM NxT Flow Cytometer (ThermoFisher Scientific). Cell cultures were diluted 1000x in deionised water and 100 μL cells were analysed at a speed of 12.5 µL/min. The scatter signal was recorded in logarithmic scale. Threshold values for forward scatter and side scatter were set at 1000 and 300, respectively to eliminate background signals from debris. The signal was gated using forward and side scatter to exclude non-singlet cells. The cytograms was drawn using Attune NxT Flow Cytometer software version 3 and edited using Inkscape.
Leica HCX PL FLUOTAR oil objective lens on a Leica DM6000 B microscope. Images were acquired by Leica Application Suite X software. Image analysis was carried out by ImagJ software. Flow cytometry analysis was carried out with Attune TM NxT Flow Cytometer (ThermoFisher Scientific). Cell cultures were diluted 1000x in deionised water and 100 μL cells were analysed at a speed of 12.5 µL/min. The scatter signal was recorded in logarithmic scale. Threshold values for forward scatter and side scatter were set at 1000 and 300, respectively to eliminate background signals from debris. The signal was gated using forward and side scatter to exclude non-singlet cells. The cytograms was drawn using Attune NxT Flow Cytometer software version 3 and edited using Inkscape.
10
Transmitted-Light STED-CW Confocal Microscopy
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A Leica TCS SP5 STED-CW scanning confocal microscope (Leica Microsystems, D) has been employed for transmitted-light imaging. The laser source consists in a 633 nm He-Ne beam (power P ~ 10 μW on the sample plane), which is focused on the sample by a 20 × 0.5-N.A. air objective (HCX PL Fluotar, Leica Microsystems, D). Images have been acquired by detecting the transmitted light signal with a non-spectral dedicated photo-multiplier tube, with no confocal pinhole along the detection optical path. A 400-Hz raster scan frequency per line has been adopted, and millimeter-sized sample regions have been imaged using a tile-scan acquisition mode.
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