Af546 conjugated and af680 conjugated secondary antibodies

Femurs were dissected, fixed in 4% paraformaldehyde and decalcified in 0.5 M EDTA at 4 °C for 2 weeks. Femurs were then transferred to 30% sucrose–PBS at 4 °C for 2 d, incubated in equal-volume OCT-30% sucrose overnight and embedded in OCT (Sakura Finetek). For collagen staining, sections were incubated with primary antibodies against collagen I (AB765P, Sigma-Aldrich), collagen type III (ab7778, Abcam) or collagen type IV (AB756P, Sigma-Aldrich), followed by treatment with peroxidase-conjugated anti-rabbit antibody (ab205718, Abcam) and 3,3’-diaminobenzidine (D8001, Sigma-Aldrich). Femur sections were stained with antibodies against EMCN (AF4666, Novus Biologicals) and laminin (ab11575, Abcam, both at 1:200). Primary antibodies were labeled with AF546-conjugated and AF680-conjugated secondary antibodies (Thermo Fisher Scientific). Musculus quadriceps femoris samples were stained with anti-mouse CD31 antibody (MEC13.3, BD Biosciences). Fluorescent images were acquired with a Nikon 80i microscope (Nikon). Light microscopy images were captured using a digital slide scanner, NanoZoomer 2.0RS (Hamamatsu), and analyzed using ImageJ software.