Bz x series microscope

All imaging was conducted on an “All in One” BZ-X series Keyence microscope and analyzed using the BZ-X Keyence Analysis software (Keyence, Osaka, Japan).

Subset of five wild type and five QPRT^−/− mice per age group were euthanized and perfused with PBS before muscle extraction. Immediately after tissue collection, samples were weighed, snap‐frozen in dry‐ice‐cooled isopentane and stored at −80°C for subsequent morphological analysis. Staining procedures were followed as previously described (Ward et al., 2018 (link)). Cryosections were cut from the middle of the gastrocnemius and tibialis anterior muscles, air‐dried to a glass microscope slide, fixed with 4% paraformaldehyde, and labeled with Alexa‐488–conjugated wheat germ agglutinin (WGA; 1 mg/mL at 1:5000 in 1× PBS, Thermo Fisher Scientific W11261) to illuminate each myofibril. Muscle sections were imaged under 20× objective, high‐resolution wide‐field introverted GFP fluorescence BZ‐X Series Keyence microscope (Keyence Corporation of America). Cross‐sectional areas of each muscle fiber were measured using the Myosight plugin for ImageJ and Fiji software.

HBMEC, HIMEC and HKMEC endothelial cells, were cultured in an 8 well pretreated chamber slide until confluence. For assays with proinflammatory pre-stimulation, confluent cell monolayers were stimulated with 10 ng/ml TNF-α (Sigma, T0157) for 24 hours at 5% CO₂ and 37°C. Cells were fixed with 3.7% paraformaldehyde for 30 min. For antibody labeling, cells were pretreated with background blocking agent (Background Buster, Innovex Biosciences, NB306) for 30 min. Cells were then washed with 1x PBS and incubated with primary mouse-anti-human-ICAM-1 (1:200 dilution) in PBS (supplemented with 2% BSA) for 1 hour (Biosource International, ThermoFisher Scientific, clone C14), followed by the secondary antibody goat anti-mouse Alexa Fluor 488 (1:200 dilution) (Invitrogen) for 1 hour. Images taken under 400x magnification (Keyence BZ-X series microscope).