3.6. Evans blue dye extravasation

Evans blue dye (EBD) is an auto-fluorescent, non-toxic compound that can be safely given to mdx mice in order to determine the extent of skeletal muscle cell damage and myofiber permeability at the microscopic level [23].

EBD is injected intraperitoneally at a dose of 1% volume to gram body weight (e.g. 250 μL for a 25 g mouse) 16 hours prior to harvesting the muscle [24,25].

Desired muscles (we use quadriceps and gastrocnemius) should be isolated and freshly frozen in OCT for creating 6 μm frozen sections.

Once the sections have been mounted on coverslips, tissue can be analyzed for dye extravasation into the myocytes by fluorescence microscopy and semi-quantitative imaging.

Alternatively, anatomically matched whole muscle tissue can be processed for total Evan’s blue dye content (via spectrophotometric absorbance at 620 nm) normalized to input muscle mass as previously published [26].