Nuclear dye 4 6 diamidino 2 phenylindole dapi

mTSPC were grown on 20 μg/mL collagen type I-coated slides and fixed with 4% paraformaldehyde. After permeabilization and blocking with 2% bovine serum albumin/phosphate buffered saline (PBS), cells were incubated overnight at 4°C with primary antibodies against CD146 (Millipore), CD105 (R&D Systems), CD90.2, CD73, and CD44 (all Novus Biologicals), Sca-1 (Abcam), Nestin (Proteintech), Nanog (R&D Systems), Scx (Abcam), and Tnmd [3 (link)]. Then, corresponding Alexa Fluor 488-conjugated secondary antibodies and nuclear dye 4′,6-diamidino-2-phenylindole (DAPI; both Life Technology) were applied at room temperature. Photomicrographs were taken on the Observer Z1 microscope equipped with the Axiocam MRm camera (Carl Zeiss). Immunofluorescence experiments were reproduced twice or thrice independently and representative images are shown.

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.