Evans Blue dye (EBD) was used as an in vivo marker of myofiber damage (Hamer et al., 2002; Salimena et al., 2004). Briefly, 100 ml EBD (Sigma, MO, USA) dissolved in phosphate-buffered saline (PBS; 0.15 M NaCl, 10 mM phosphate buffer, pH 7,0), sterilized by filtration through 0.2 mm membrane

(Millipore Corp, MA, USA) then injected intraperitoneally (1 mg EBD/10 g body weight). Mice were killed 24 h later, muscles were snap frozen in OCT (Tissue-TEK; Elkhard, IN, USA), and 5 μm thick frozen sections fixed in acetone for 2 min, air-dried, quick-dipped in xylene and GDC-0980 supplier mounted with Enthelam (Merck, Damstadt, German). High definition whole area images of all cross-sections from each mouse at a time point were obtained from individual photomicrographs with a microdigital camera mounted on a Zeiss Axioplan microscope (Zeiss, Oberkochen, Germany) using a 10× objective and observed under bright field and fluorescence optics. Gastrocnemius muscles were embedded in OCT (Tissue-TEK; Dasatinib supplier Elkhard, IN, USA) and frozen in isopentane alcohol submerged in liquid nitrogen. 5 μm thick frozen sections were placed on poly-l-lysine (Sigma, St. Louis, Missouri) pre-coated slides and allowed to dry at room temperature for 4 h before staining. Hematoxylin-eosin

(Merck, Darmstadt, Germany) was used to verify morphological alterations and syrius red staining to detect collagen deposition. Captured images from three different levels of all cross-sections at each time point were acquired with a microdigital camera mounted on a Zeiss Axioplan microscope (Zeiss, Oberkochen, Germany) using a 20× objective. Images were mounted with Photomerge Adobe Photoshop CS3 software. Total surface area and areas occupied by injury and collagen deposition were determined with Image-Pro 4.5 (Media Cybernetics, Inc.). Results are expressed as percentage of total area in each cross-section. 5 μm,

spaced 500 μm cryostat cross-sections were mounted on poly-l-lysine pre-coated slides, fixed in acetone (−20 °C), blocked for endogenous peroxidase activity with 3% hydrogen peroxide in PBS for 30 min, and for unspecific antigens with PBS containing 5% of goat serum. Sections were then incubated Oxymatrine at room temperature for 60 min with primary monoclonal rat IgG anti-F4/80 (clone CI; A3-1; Serotec, Oxford, UK) at a 1:50 dilution in phosphate-buffered saline (PBS) followed by incubation with streptavidin–peroxidase complex (1:300; Sigma) and further washed with PBS. Enzyme activity was revealed with aminoethyl-carbazole (Sigma) in the presence of hydrogen peroxide. All sections were lightly counter-stained with Mayer’s hematoxylin (Sigma). Percentage of F4/80 positive areas in the injury foci was determined with Image-pro Plus 4.5 software (Media Cybernetics Inc., Silver Spring, MD). It included three mice per experimental group and analyzed six frozen sections per animal.