Anti cd11b

Harvested ears were treated with a depilatory reagent (Nair), washed in a solution of phosphate buffered saline and Triton-X 100 (Sigma-Aldrich), followed by fixation in 4% PFA (Affymetrix) at 4 °C. The cartilage was then removed and the ear was split to expose the subcutaneous tissues. Sections were blocked in secondary serum and incubated in primary antibody solutions (anti-podoplanin (Abcam), anti-CD11b (R&D Systems), and anti-iNOS (BD Biosciences) overnight at 4 °C followed by fluorescent-conjugated secondary antibodies (Life Technologies).

From both treated and untreated mdx mice DIA, QA and TA muscles were removed, frozen in liquid nitrogen-cooled isopentane, and sectioned on cryostat. We performed histological analysis on QA and TA as these muscles show similar level of dystrophic degeneration in mdx mice. Furthermore we investigated DIA muscle as it is the muscle that mostly recapitulates human pathology. Serial sections of 10 μm in thickness were stained with H&E and AM. Images were captured with LEICA AS LMD optical microscope. For immunofluorescence analysis, sections were incubated with antibodies: rabbit anti-laminin (1 : 50; Abcam), rat anti-CD31 (1 : 100; BD), anti-α smooth muscle actin (1 : 50 Sigma), and anti-CD11b (1 : 50, R&D). The slides were analysed using a fluorescent microscope (LEICA DMIRE2) and a confocal microscope (LEICA TCS-SP2). For quantitative analysis ImageJ Software (NIH) was used. For CD11b and DHE correlation study serial sections were utilised.