Tissue specimens were fixed in 4% neutral buffered formaldehyde solution for 48 h, decalcified by Biodec R (Bio-Optica, Milan, Italy) for 6 h and then routinely processed for paraffin embedding. Six-μm-thick paraffin-embedded tissue sections were deparaffinized and rehydrated with xylene and a graded series of ethyl alcohols, and stained by Haematoxylin and Eosin (H&E), Toluidine Blue or Safranin O.
For immunohistochemistry, deparaffinized and rehydrated slides were incubated with 3% hydrogen peroxide for 30 min to block endogenous peroxidase activity. Antigen retrieval was performed in Citrate buffer pH 6 at 70°C for 10 min (BMP-2 and Decorin) and in 0.3% Tween 20 in 1x PBS at RT for 20 min (TGF-β). Sections were then incubated overnight at 4°C with anti- BMP-2 (ab14933, Abcam, Cambridge, UK; 1:250), anti-TGF-β*****(GTX21279, GeneTex, Irvine, CA, USA; 1:200) or anti-decorin (ab175404, Abcam; 1:400) antibodies. Antigens were visualized by Envision Dako REAL™ EnVision™ Detection System (K5007, Dako, Santa Clara, CA, USA). Sections were counterstained with Mayer’s haematoxylin, dehydrated, and mounted in Biomount HM (Bio-Optica). Healthy bone was used as positive control. Omission of primary antibody and muscle biopsies were used as negative controls.
For immunohistochemistry, deparaffinized and rehydrated slides were incubated with 3% hydrogen peroxide for 30 min to block endogenous peroxidase activity. Antigen retrieval was performed in Citrate buffer pH 6 at 70°C for 10 min (BMP-2 and Decorin) and in 0.3% Tween 20 in 1x PBS at RT for 20 min (TGF-β). Sections were then incubated overnight at 4°C with anti- BMP-2 (ab14933, Abcam, Cambridge, UK; 1:250), anti-TGF-β*****(GTX21279, GeneTex, Irvine, CA, USA; 1:200) or anti-decorin (ab175404, Abcam; 1:400) antibodies. Antigens were visualized by Envision Dako REAL™ EnVision™ Detection System (K5007, Dako, Santa Clara, CA, USA). Sections were counterstained with Mayer’s haematoxylin, dehydrated, and mounted in Biomount HM (Bio-Optica). Healthy bone was used as positive control. Omission of primary antibody and muscle biopsies were used as negative controls.