Histochemical analyses for muscle lesion and immunohistochemical analyses for collagen type I and type III. (A) Hematoxylin-eosin, (B) Masson trichrome and (C) picrosirius staining of the boundary between the contraction band (CB) and unaffected adjacent muscle (AM). Arrows indicate muscles that are undergoing fibrosis. (D) picrosirius staining of the boundary viewed under polarized light microscopy (collagen type I is yellow and type III is green). (E, F, G, H) Immunohistochemical staining of contraction band (E, G) or unaffected adjacent muscle (F, H) was carried out with antibodies against collagen type I (E, F) or collagen type III (G, H). Proteins specifically recognized by the antibodies are stained red, whereas nuclei are counter-stained blue. Scale bars: 100 μm (A, B, C, D), 50 μm (E, F, G, H).