MIA Induced TMJOA in Rat Figure 1. Outline of experimental design and confirmation of injection site into upper compartment of rat TMJ. A: Outline of experimental design. B: Photograph of dye (fast green solution) injection into the upper compartment of the left TMJ. (a). Needle insertion was 5 mm anterior to the external auditory canal. (b). Dissection showed that the needle was right under the root of the zygoma (dotted arrow), anterior of the external auditory canal (arrow), stopped at the temporal fossa, and was located in the upper compartment (black circle). (c). 50 mL dye was injected. (d). Opening the capsule revealed that the dye was restricted to the upper compartment of the TMJ (disc and condyle: hollow arrow). doi:10.1371/journal.pone.0045036.g001 Caspase8, and Caspase9 [37], and alfa-smooth muscle actin (a- SMA) [38] were all previously described and their efficiency was confirmed by sequencing their conventional PCR products. The primers for rat Matrix Metalloproteinase (MMP)3 (sense: 59- ACCTATTCCTGGTTGCTG-39; anti-sense: 59- GGTCTGTGGAGGACTTGTA-39), MMP13 (sense: 59- CTGACCTGG- GATTTCCAAAA-39; anti-sense: 59- ACACGTGGTTCCCTGAGAAG-39), TIMP1 (sense: 59- CCTCTGGCATCCTCTTGT-39; anti-sense: 59-TTGATCT- CATAACGC- TGGT-39), and Proliferating Cell Nuclear Antigen (PCNA) (sense: 59-CCAGGG- CTCCATCCTGAA-39; anti-sense: 59-CCCAGCAGGCCTCATTGAT-39) were designed with Prim- er Premier Version 5.0 software and their efficiency was confirmed by sequencing their conventional PCR products. Terminal Deoxynucleotidyl Transferase dUTP nick end Labeling (TUNEL) Assay Apoptosis was examined in situ using a TUNEL assay according to the manufacturer’s instructions (Roche, Mannheim, Germany). Briefly, sections were deparaffinized, rehydrated, pretreated with protease K (10 mg/ml, Sigma) for 20 min, and blocked with 3% bovine serum albumin for 20 min at room temperature. The sections were incubated with TUNEL reaction mixture for 1 h at 37uC and covered with fluorescence mounting medium (Zhong- shan-Golden-Bridge-Biotechnology, Beijing). Confocal microscop- ic images were acquired using a Zeiss laser-scanning microscope (LSM 510). PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e45036