Quantifying Mitochondrial Biomarkers in Tendon Tissue

The protein expression of biomarkers to assess mitochondrial status in the tendon tissue sections was analyzed by immunostaining following our previously reported protocols^{29 (link),38 (link)}. Briefly, the sections were subjected to antigen retrieval by heating at 95 °C for 20 min in HIER buffer (Heat Induced Antigen Retrieval) (TA-135-HBM) followed by the use of blocking solution (0.25% Triton X-100 and 5% horse serum in PBS) at room temperature for 2 hrs. Primary antibodies against Bcl2 (sc-7382, Santa Cruz Biotechnology, Inc), BAX (ab-32503, Abcam), citrate synthase (ab-96600, Abcam), complex-1 and PGC-1α (sc-518025, Santa Cruz Biotechnology, Inc) were used in a dilution of 1:50. Corresponding fluorochrome-conjugated secondary antibodies with a dilution of 1:200 were used to bind the primary antibodies. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (H-1200) and imaged using a fluorescent microscope (Olympus BX51; Olympus America, Center Valley, PA). The fluorescence intensity was quantified using ImageJ software and the results are expressed as mean fluorescence intensity (MFI). A negative control with secondary antibodies alone was maintained in a similar manner to detect background fluorescence and to fix the exposure time.