Lumican

Human biopsies from Achilles tendon (grant No.: 166-08) as well as mouse whole foot were embedded in paraffin and cut in 6 μm thick longitudinal sections. Prior to staining, slides were deparaffinated with xylol and rehydrated via ethanol (100–50% EtOH). For antigen retrieval, sections were treated with 0,2% hyaluronidase for 30 min. Following blocking with 1% BSA/PBS for 2 h sections were incubated overnight at 4 °C with primary antibodies against asporin, fibromodulin, lysyl oxidase (All Abcam), lumican (Santa Cruz, Dallas, USA) and Tnmd (Docheva et al., 2005 (link)). Corresponding Alexa Fluor 488-labeled secondary antibodies and nuclear dye 4′, 6-diamidino-2-phenylindole (DAPI) (both Life technology, Carlsbad, USA) were applied at room temperature for 1 h and 5 min, respectively. Photomicrographs were taken with an Axiocam MRm camera on Observer Z1 microscope (Carl Zeiss, Oberkochen, Germany). Confocal photomicrographs of Tnmd staining in human and mouse Achilles tendon were taken using a confocal Leica TSC SP2 microscope (Leica, Wetzlar, Germany). Immunofluorescence experiments were repeated thrice independently.