Evos xl digital inverted microscope

The cells (0.3 ~1 × 10⁴ per well) in the medium containing low serum (1%) were seeded into 24-well plates by adding the cell suspension to 6.5-mm upper chambers of transwells with 8-mm pores (BD Biosciences, Franklin Lakes, NJ, USA) that had been pre-coated for 2 h with Matrigel (BD Biosciences, Bedford, MA, USA) in a 37 °C incubator. Meanwhile, 0.65 mL of 20% FBS complete medium, with or without different concentrations of compounds, was added to the lower chamber as a chemoattractant. After 48 h, the invading cells that had not migrated through the filter in the transwell inserts (on the upper surface) were removed with a cotton swab, and the cells that had migrated to the lower chamber were fixed in paraformaldehyde and stained with a 0.1% crystal violet solution. Four random fields were selected and photographed under an EVOS XL digital inverted microscope (Life Technologies, Carlsbad, CA, USA) at a magnification of 200×.

Quadriceps were collected and frozen in 2-methyl-butane cooled under liquid nitrogen. Quadricep samples were sectioned using a CryoStar NX350 HOVP Cryostat (Thermo Scientific, Waltham, MA, USA) at −20 °C with a thickness of 10 μm through the mid-belly and mounted on SuperFrost glass slides (Electron Microscopy Sciences, Hatfield, PA, USA, catalog no. 71882-01). For analysis of fiber cross-sectional areas (CSAs), fibers were assessed by hematoxylin and eosin (H&E staining), while for fiber type, muscles were stained using NADH/NBT (NADH and nitro blue tetrazolium) staining as described in [25 (link),45 (link)]. Light-stained fibers were labeled as Type IIb fibers, medium-stained fibers as Type IIa, and dark-stained as Type I fibers. The images were taken using a 20× objective of an EVOS XL digital inverted microscope (Life Technologies, Carlsbad, CA, USA). Muscle fibers were individually counted in each image by a blinded investigator, and the cross-sectional areas were measured by outlining 150 randomly chosen fibers per image and using ImageJ [46 (link)].