Animals were euthanized with anesthetic overdose. Achilles tendons were isolated from surrounding tissue and dissected from both sides of hind limbs. The proximal ends of the Achilles tendons were disconnected at the end of the gastrocnemius, plantaris, and soleus, and the distal ends at the calcaneus. Left leg tendons of eight control rats and six burn rats for each time point (1, 3, 7, and 14 days) were stored in RNAlater (Qiagen), whereas right leg tendons were snap frozen for protein extraction. Samples were stored in −80°C for further analysis. Tendon RNA was obtained with RNAeasy Universal kit (Qiagen). cDNA conversion was done with iScript kit (Bio-Rad), and qPCR with SsoFast Eva Green kit (Bio-Rad). Primers for IL-6, TNF, IL-1β, col1a1, col3a1, MMP9, MMP13, and TGFβ1 were purchased from QuantiTect Primers (Qiagen). Gene expression was calculated using the ΔΔCt method with 18 s as housekeeping.
For protein extraction, tendon tissue was homogenized in RIPA buffer (Invitrogen) with proteinase inhibitor cocktail (Sigma). Protein quantification was performed with BCA assay (Pierce); 10 μg of total protein was used for Western blot. Antibodies included anti-collagen I (Abcam), anti-collagen III (Abcam), goat anti-mouse HRP (Pierce), and goat anti-rabbit HRP (Pierce), which were prepared in 2% BSA. Blots were developed with ECL (Pierce) using a CCD camera system.
For protein extraction, tendon tissue was homogenized in RIPA buffer (Invitrogen) with proteinase inhibitor cocktail (Sigma). Protein quantification was performed with BCA assay (Pierce); 10 μg of total protein was used for Western blot. Antibodies included anti-collagen I (Abcam), anti-collagen III (Abcam), goat anti-mouse HRP (Pierce), and goat anti-rabbit HRP (Pierce), which were prepared in 2% BSA. Blots were developed with ECL (Pierce) using a CCD camera system.