Sc 87425

The cells (passage 2) were grown in a chamber slide to 70% confluence in DMEM. The media was then removed, washed in sterile PBS, immediately fixed with 5% formalin for 30 min and immunofluorescence assay for tendon specific biomarkers including tenomodulin and scleraxis were carried out by following the above-mentioned protocol. The primary antibodies used were anti-goat tenomodulin (sc-49324, Santa Cruz Biotechnology, Inc) and anti-goat scleraxis (sc-87425, Santa Cruz Biotechnology, Inc), and the secondary antibody used was donkey-anti-goat-594. The images were acquired using a fluorescent slide scanner system (VS120-S6-W, Olympus) at 20x magnification and the images were taken and converted to TIF format using OlyVIA Desktop software. DAPI was used to counterstain the nuclei.

The regenerated patellar tendon tissues were washed with PBS, fixed in buffered formalin, embedded in paraffin and sectioned for histological examination. Immunohistochemistry was done as described previously [26 (link)]. Briefly, after deparaffination, the sections were rehydrated, quenched of endogenous peroxidase activity and subject to antigen retrieval. After blocking with 5% normal donkey and goat serum, the sections were incubated with specific antibodies against Tnmd (sc-98875, Santa Cruz Biotechnology), Collagen I (AF7001, Affbiotech), GDF6 (bs-11843R, Bioss), Egr1 (bs-1076R, Bioss), OPN (bs-0026R, Bioss), OCN (bs-0470R, Bioss), MMP13(bs-0575R, Bioss) and Scx (sc-87425, Santa Cruz Biotechnology) at dilution of 1:200 at 4 °C overnight. Goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody and donkey anti-goat horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology; dilution 1:200) were then added for 30 min, respectively. Afterward, the sections were rinsed, counterstained in hematoxylin or methylgreen, dehydrated with graded ethanol and xylene, and mounted with p-xylene-bis-pyridinium bromide (DPX) permount (Sigma Aldrich, St Louis, MO, USA). All incubation times and conditions were strictly controlled. The sections were examined under light microscopy (DMRXA2, Leica, Germany).

The formed neo-tendon tissue and regenerated patellar tendon tissue were washed in PBS, fixed in buffered formalin and 70% ethanol, embedded in paraffin and sectioned for staining with hematoxylin and eosin. Immunohistochemistry was done as described previously [39 (link)]. Briefly, after deparaffination, the sections were rehydrated, quenched of endogenous peroxidase activity and subject to antigen retrieval. After blocking with 5% normal donkey and goat serum, the sections were incubated with specific antibodies against Tnmd and Scx (sc-98875 and sc-87425, Santa Cruz Biotechnology, CA, USA) at dilution of 1:100 at 4°C overnight. Goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody and donkey anti-goat horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology, CA, USA; dilution 1:100) were then added for 30min respectively. Afterward, the sections were rinsed, counterstained in hematoxylin, dehydrated with graded ethanol and xylene, and mounted with p-xylene-bis-pyridinium bromide (DPX) permount (Sigma Aldrich, St Louis, MO, USA). All incubation times and conditions were strictly controlled. The sections were examined under light microscopy (DMRXA2, Leica Microsystems Wetzlar GmbH, Germany).