Ez suite software

Sections of trunk muscle directly posterior to the dorsal fin were collected (4–8 fish per tank; n=7 tanks per exposure group) and fixed in 10% neutral-buffered formalin for 18 hours, progressively dehydrated in ethanol, and paraffin-embedded. Blocks were cut into 7 μm sections and one muscle trunk sample per tank at 16 weeks was used for hematoxylin and eosin (H&E) staining (n=6 fish per exposure group). Once the fish could be sexed, approximately 3 males and 3 females per tank were taken for muscle fiber density quantification at 28, 40 and 52 weeks. Slides were imaged using a Leica ICC50 HD light microscope and Leica EZ suite software. ImageJ software (National Institutes of Health) was used to count muscle fibers. Upper right quadrants of each tissue section were selected for quantification and areas were determined (mm²) to calculate density (fibers/mm²). Average densities for all fish and males/females were determined for each tank.

Muscle tissue samples were fixed in 10% neutral buffered formalin for 18 h, progressively dehydrated in ethanol, and embedded in paraffin. Blocks were cut into 7μm sections and slides were stained using Masson’s trichrome collagen staining kit (Richard-Allan Scientific; Kalamazoo, MI). Slides were imaged using a Leica DM750 light microscope and Leica EZ suite software. ImageJ software (National Institutes of Health) was used to quantify the amount of collagen present in the injury site. Using the circle tool, a 1.5 mm × 1.5 mm circle was placed over the injury site. After converting the image to an RGB (red, green, blue) stack and selecting the “red” channel, the threshold function was used to isolate the area of collagen. Percent area of collagen within the circle was then calculated using the “Measure” function. Average percent areas for day 3 and 10 post-injury samples were calculated for each exposure group (n=7) and baseline muscle samples (day 0) were calculated for each exposure group (n=3 per sex per tank) at 28, 40 and 52 weeks of age. Cardiotoxin injection was not done at 16 weeks due to the small size of the fish.