8-Isoprostane levels (lipid peroxidation index) were measured in cell lysates using the 8-isoprostane EIA kit (Cayman Chemical Co., Ann Arbor, MI, USA), following the manufacturer’s instructions. Moreover, lipid peroxidation was investigated by confocal microscopy using BODIPY 581/591 C11 (Life Technologies, Carlsbad, CA, USA) [69 (link)]. The fluorescent probe BODIPY 581/591 C11 shifts its fluorescence from red to green in the presence of oxidizing agents. Fluorescence was detected using a confocal Leica TCS SP5 scanning microscope (Mannheim, Germany) using a Leica Plan Apo 63X oil immersion objective and then projected as a single composite image by superimposition. Lipid peroxidation was also quantified by FACS analysis. Cells were incubated in DMEM with BODIPY 581/591 C11 (2 μM) for 30 min at 37 °C [70 (link)] and analyzed using a FACSCanto flow cytometer (Becton-Dickinson, San Jose, CA, USA).
Plan apo 63x
Manufactured by Leica
Sourced in Germany
The Plan Apo 63X is a high-performance microscope objective lens developed by Leica. It features a numerical aperture of 1.40 and is designed for use with oil immersion. The lens provides a flat image field and excellent chromatic correction, making it suitable for a variety of scientific and research applications.
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2 protocols using plan apo 63x
1
Quantitative Analysis of Lipid Peroxidation
2
Confocal Imaging of Fluorescent Cells
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Images of immunostained cells or cells expressing fluorescent tagged proteins were acquired on Confocal inverted microscope SP8-X (DMI 6000 Leica). Optical sections were acquired with a Plan Apo 63x oil immersion objective (N.A. 1.4, Leica)
using the LAS-X software. Fluorescence was excited using either a 405nm laser diode or a white light laser, and later collected after adjusting the spectral windows with GaAsP PMTs or Hybrid detectors. Images from a mid-focal plane are shown. For Huh7 experiments, images were acquired by the ZEISS LSM800 Airyscan.
using the LAS-X software. Fluorescence was excited using either a 405nm laser diode or a white light laser, and later collected after adjusting the spectral windows with GaAsP PMTs or Hybrid detectors. Images from a mid-focal plane are shown. For Huh7 experiments, images were acquired by the ZEISS LSM800 Airyscan.
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