Amg evos fl microscope

Histological examination was carried out as described previously [25 (link)]. In brief, TA muscles were collected at various time points and preserved for one day in formaldehyde (4% in PBS). Before embedding in paraffin, the tissue was dehydrated as per standard protocol. After the paraffin solidified, the blocks were cut into 6µm thick sections using a microtome. Hematoxylin and eosin (H&E) staining was used after deparaffinization to determine overall morphology and the presence of necrotic fibers following damage. An AMG EVOS fl microscope was used to photograph the sections (Thermo Fisher Scientific, Waltham, MA, USA).

Cells were seeded in 96-well plates (1500 cells/well) overnight and treated with 100 µl of IL-6 (25 ng/ml) for 24 h (37 °C). EdU assays (Beyotime Institute of Biotechnology) were performed according to the manufacturer’s protocol. First, 2× EdU working solution was prepared and preheated to 37 °C. A total of 100 µl of solution was added to each well of a 96-well plate for a final concentration of 10 µM. Then, the plate was incubated in the culture chamber for an additional 2 h. After the culture medium was discarded, 100 µl of 4% paraformaldehyde (precooled to 4 °C) was added to each well, and the cells were fixed at room temperature for 15 min. After removing the fixative, 100 µl of PBS was added to each well to wash the cells 3 times for 3 min each time. After the washing solution was removed, 100 µl of PBS containing 0.3% Triton X-100 was added to each well, and the cells were incubated at room temperature for 10 min. The permeabilization solution was discarded, and 100 µl of PBS was added to each well to wash the cells 3 times for 3 min each time. Images were captured using an AMG EVOS FL microscope (Thermo Fisher Scientific, Inc.) at a magnification of 200×. The average optical density was determined from three randomly selected images using ImageJ software (version 1.53a, National Institutes of Health).

Cell migration was evaluated by a scratch wound assay. The cells were seeded at a concentration of 12 × 10⁴ cells/well in 6-well plates and with SCR control or siNEDD9 siRNAs for 24 h. “Wound” scratch was performed 24 h post-transfection. In order to avoid cell proliferation during cell migration, transfection was performed in serum-starved conditions. A scratch was created with a 10 μL plastic pipette tip on the cell monolayer. The images of the scratch wounds were captured using an AMG Evos FL microscope (Thermo Fisher Scientific, Foster City, CA, USA) at 0, 3, 6, and 24 h after scratching. The migration is represented as percentage of wound closure between the time zero and respective timepoint. Area measurements were done using NIH ImageJ Imaging Software (Rayne Rasband, National Institutes of Health, Bethesda, MD, USA). Assay was performed in 3 biological repeats.