Leica dmi8 microscope

At 14 days of adipogenic differentiation, Oil Red O (ORO) staining was performed to quantify the accumulation of intracellular lipids, mainly triglycerides, in mature adipocytes obtained from all controls and under different experimental conditions (0.1, 1, and 10 µM with or without ICI 182,780 at 100 nM). Briefly, cells were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde (Electron Microscopy Science Hartfield, PA, USA) for 1 h at room temperature. After washing with milliQ-water and 60% isopropanol, cells were stained with a filtered ORO solution (0.5%, w/v) in milliQ- water (60/40, v/v) for 45 min, followed by washing with 60% isopropanol and again with milliQ-water. Cells were first observed and photographed under a Leica DMi8 microscope (Leica Microsystems, Wetzlar, Germany) with the Leica Application Suite (LAS) X software, and the retained dye was extracted with 100% isopropanol, measuring the optical density at a wavelength of 520 nm with a microplate reader (BioTek HTX, Fisher Scientific, Waltham, MA, USA) [48 (link)]. All aforementioned products were purchased from Thermo Fisher Scientific (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) or Sigma Aldrich (Sigma-Aldrich, St. Louis, MO, USA).

A549 cells were cultured in a 24-well plate (Thermo Fisher Scientific) and tethered with MYR-coreSA as described earlier. Various concentrations (0, 0.625, 1.25, 2.5, 5, 10, 15, and 20 µM) of sinigrin were added and viability after 48 h was quantified using MTT assay. The A549 cells were directly treated with the same concentrations of AITC dissolved in DMSO. The phase-contrast images were also taken after 48 h with a Leica DMi8 microscope equipped with a Leica EC3 camera (Leica Microsystems) to observe the cell morphological changes.

Stem samples were harvested from the 20th internode and fixed in FAA solution (50% ethanol, 10% formaldehyde, and 5% acetic acid). Trimmed stem segments were dehydrated in a graded ethanol series (50%, 60%, 80%, 90%, and 100%), and were embedded in LR White Hard resin (TAAB, Aldermaston, UK) with 5% PEG400. Cross Sections (2-µm thick) were cut using a rotary microtome (RX-860; Yamato Kohki Industrial, Saitama, Japan) and then stained with a 0.5% (w/v) toluidine blue solution. Sections were imaged using a Leica DMi8 microscope with a Leica DFC7000T microscope camera (Leica Microsystems, Wetzlar, Germany). Cell wall thickness and cell size of wood fiber cells were measured using the ImageJ software (n = 100 for each plant)^{59 (link),60 (link)}.