Eclipse te300

Saos-2 cells were seeded in 6-well plates under normal culture conditions at the density of 5.0 × 10⁵ cells/well. Once it reached ~90% confluence, the medium was removed and the cell monolayer was scratched across the center using a sterile pipet tip (200 μL) to produce a uniform wound area. Through gently washing with sterile PBS1X, cellular debris was removed from the well and the cells incubated with 48 μM of polydatin. Cell migration was monitored by microscopy using an inverted light microscope (Eclipse TE 300 Nikon, New York, USA), and images were captured (10x magnification) at different time points after the scratch (0–48 h). The images acquired were analyzed quantitatively, using a specific wound healing tool of ImageJ software (Maryland, USA). The percentage of relative wound closure was assessed according to the following equation [23 (link)]:
$\begin{array}{c}Woundclosure\%=\frac{W_0-W_n}{W_0}\times 100,\end{array}$ in which W_n is the width of the cut after the treatment time and W₀ is the width of the scratch before treatment.

Liver tissue was harvested at ZT 6 (12 p.m.) following fasting from ZT 0 (6 a.m.) on the final day of the experiment. Hepatocellular steatosis was measured by Oil Red O staining on 7 μm cryosections of liver tissue [30 (link)]. Images were scanned by bright-field light microscopy (Eclipse TE300, Nikon, NY, USA) at ×20 magnification. The area of Oil Red O lipid staining was measured using Image J software (Version 1.54g) [47 (link)]. Hepatic steatosis was further assessed by hematoxylin and eosin (H&E) staining of 5 μm liver tissue sections of paraffin-embedded liver tissue. Images were scanned by a Nanozoomer 2.0HT Slide Scanner (Hamamatsu Photonics, Bridgewater, NJ, USA), and the number and area of lipid droplets (stained negative for H&E) were measured using Image J. Hepatic inflammation was assessed by counting infiltrating immune cells in the H&E-stained sections. Inflammation scoring was performed by a trained pathologist blinded to the sample identities. Liver fibrosis was assessed by Sirius Red staining and quantified by Image J.

Fluorescence was visualized using an inverted microscope (Nikon Eclipse TE300, Nikon, Tokyo, Japan). After selecting a 510/20-nm bandpass filter for EGFP detection, EGFP-positive mice were identified.

Immunofluorescence analysis was performed as standard procedures. In brief, NRK cells or primary mouse cortical neurons at DIV9 on glass coverslips were washed twice in PBS, fixed in 4% paraformaldehyde/PBS at room temperature for 15 min, incubated with 50 mM NH₄Cl to quench free aldehyde groups, washed twice in PBS and labeled with primary antibodies against pan-kalirin (polyclonal against the 4 to 7 spectrin repeats of kalirin; EMD Millipore) and Htt (MAB2166). Targeted proteins were visualized by incubation of cells on glass coverslips with secondary antibodies conjugated with BODIPY or Cy-3 (Jackson Laboratory). Microscopy was performed using a 60x or 100x oil Nikon Plan Apo objective lens mounted on an inverted Nikon Eclipse TE300 fluorescent microscope. Images were collected using a Bio-Rad Laser-sharp confocal system equipped with krypton-argon and blue diode lasers, and merged using PhotoShop.

Formalin eWAT samples were embedded in paraffin and 5 µm sections made with a microtome (HistoCore Multicut, Leica Biosystems, Nanterre, France) were placed on glass slides for staining. To evaluate adipocyte size, the eWAT tissues were stained by hematoxylin QS (Vector Laboratories, Newark, CA, USA). The sections were mounted in mounting medium, Entelan (Merck, Saint-Quentin-Fallavier Cedex, France) and examined under a light microscope (Nikon Eclipse TE300). For each sample, quantification was performed with Image J 1.53c software on 10 photos taken at 200-fold magnification. Adipocyte size and distribution were calculated according to Parlee’s article [26 ].