Ctr 6500 microscope

Muscle sections were stained using the Sirius Red/Fast Green collagen staining kit, as per the manufacturer's protocol (Chondrex Inc, WA). Muscle sections were immersed in dye solution for 30 minutes at room temperature and excess dye was washed off with distilled water. Stained sections were then dried and mounted using DPX mounting medium (Merck, USA). The tissue sections were photographed and tiled using the Leica CTR 6500 microscope, equipped with the Leica DFC 420 camera and Image Pro Plus software (Media Cybernetics, Bethesda, MD) and a 10x objective. Representative images were taken at a higher magnification (40x objective). The extent of collagen deposition (red stain) was quantified by determining the total area that was stained red across the entire muscle cross section. The total area stained red was normalized to the total TA muscle section area and expressed as a percentage, to represent percentage fibrosis.

The same unique PVA substrate described above was used to induce formation of 3D tumor spheroids [45 (link)]. As previously described, 96-well u-bottom non-tissue culture-treated plates were coated with 0.75% (wt/vol) PVA and allowed to dry upside down. LN229 cells, obtained from ATCC, were cultured to confluence in DMEM (High Glucose DMEM (Gibco, Grand Island, NY, USA) + 5% FBS (HyClone, South Logan, UT, USA), trypsinized, and resuspended at a plating concentration of 15 × 10⁴ cells/mL, and 50 µL (7500 cells) plated per well into the internal wells of the prepared plate. To give the indicated final concentrations, 50 µL of compound (prepared in media) was added to the wells (2× replicates per compound) and 100 µL of PBS was plated per well into the external wells of the plate (to buffer edge effects), and the plate was cultured for 7 days at 37 °C and 5% CO₂. Spheres were imaged at 5× magnification using a Leica CTR6500 microscope fitted with an automated stage.

Transverse sections (10 µm) were cut from the mid-belly region of OCT-embedded TA muscle using a cryostat (Leica CM 1950, Germany) and sections were stained with Gill’s hematoxylin followed by 1% eosin (Merck; H&E). TA muscle sections were photographed and tiled using the Leica CTR 6500 microscope equipped with the Leica DFC 420 camera and Image Pro Plus software (Media Cybernetics, Bethesda, MD, USA). The CSA of 200 muscle fibers from 5 random fields (1000 fibers per section per mouse) were analyzed (n = 3 mice per treatment group).

Bead aggregation assays were performed in 48-well plates pretreated with anti-adherence rinse (Stemcell Technologies, Vancouver, CA, USA) per the manufacturer’s instructions. A bacterially expressed biotin-tagged fragment of PTPµ corresponding to aa 21-365 (PTPµ21_365avi—containing the MAM, Ig, and first FNIII repeat) was diluted into PBS + 0.01% Tween 20 to 30 ng/µL. Streptavidin MonoMag beads (1 micron diameter, Ocean NanoTech, San Diego, CA, USA) were added at a ratio of 53 ng protein/µg of beads and incubated at room temperature for 15 min. Coated beads were diluted to 6 µg beads/mL (~1.6 × 10⁷ particles/mL) in PBS + 0.01% Tween 20, and 90 µL used per well. Compounds were diluted into PBS + 0.01% Tween 20 and 90 µL added per well (2 × replicates) to give a final concentration of 100 µM. The plate was incubated at room temperature for 20 min; then, aggregation was induced by rotation at 150 rpm for 30 min. The plate was manually shaken to randomly distribute aggregates, and sixteen 20× images (a 4 × 4 grid with 1000 micron spacing in both x- and y-axes) captured per well on a Leica CTR6500 microscope fitted with an automated stage. This does not tile the entire well, but captures a sampling to account for the random distribution of particles.

Cells were seeded at a density of 7500 cells per well into the internal wells of 96-well plates coated with 0.75% (wt/vol) PVA as previously described [59 (link)]. Compounds were added (2x replicates per treatment) at the indicated final concentrations, and control wells were treated with matching concentrations of DMSO. The external wells of the plates were filled with PBS to buffer against edge effects, and the cells were incubated at 37° C and 5% CO₂ for 7 days. A Leica CTR6500 microscope fitted with an automated stage was used to capture brightfield images on day 1 and day 7, and sphere footprint areas were measured using Image J (v1.52a http://imagej.nih.gov/ij) as previously described [59 (link)]. To quantify the effects of the compounds on day 1, the footprint areas of the treated wells were normalized to the average area of the matched DMSO control wells. To quantify the effects of the compounds on sphere growth, the change in the sphere footprint areas was calculated (day1/day7*100) and then normalized to the average size change of the matched DMSO samples. All values are presented as average percentages ± s.e.m.

WSU-FSCCL cells were incubated with 40 μg/ml DC-SIGN-Fc in medium containing 2 mM Ca²⁺ for 1 h at 4 °C. Anti-IgM (20 µg/ml) was added for the last 30 min. Cells were transferred to poly-L-lysine-coated glass slides (Sigma), fixed with acetone (Sigma) and stained with mouse anti-human CD209 (Clone DCN46, BD Biosciences) at 4 °C overnight. Cells were incubated with secondary antibodies AF568 donkey anti-goat (anti-IgM detection) and AF488 donkey anti-mouse (DC-SIGN-Fc detection) (both Life Technologies). Nuclei were visualized with DAPI (Sigma), and coverslips were mounted with Mowiol (Sigma). Confocal images were collected using a Leica TCS-SP5 digital confocal system coupled to a Leica CTR6500 microscope.

After 24-week CS exposure, the grip force test was performed to evaluate the muscle strength of limbs using a grip strength meter (unibiolab, Beijing, China) as reported previously (Kehl et al., 2000 (link)). The researchers were blinded to group allocation. In brief, the mouse was grasping a mesh bar connected to a force transducer with all four limbs and was being pulled back by the tail at a constant speed until its grip was released. The peak force (g) produced during the process was recorded using the transducer. The mean of three attempts was recorded as grip strength. The cross-sectional area (CSA) of muscle fibers was measured as described previously (Chiu et al., 2018 (link)). Transversal gastrocnemius muscle sections (10 µm thick) were cut from paraffin-embedded muscle tissues using a cryostat (Leica, Solms, Germany) and stained with hematoxylin–eosin (H&E). Muscle sections were photographed and tiled using the Leica CTR 6500 microscope equipped with the Leica DFC 420 camera and Image Pro Plus software (Media Cybernetics, Maryland, United States). The CSA of muscle fibers was analyzed (n ≥ 3 mice per group).