After fixation, disc units were serially dehydrated in alcohol. Decalcification was accomplished with 12. 5% ethylenediaminetetraacetic acid followed by paraffin embedding. The tissue blocks were sectioned parallel to the endplate, and sections were deparaffi nized in xylene and rehydrated through graded EPZ5676 ethanol and stained with Alcian blue, eosin, and hematoxylin. For localizing matrix metalloproteinase 3, sec tions were incubated with rabbit monoclonal MMP 3 antibody in 2% bovine serum albumin in PBS at a dilution of 1,250 at 4 C overnight. After washing of the sections, the bound primary antibody was incubated with Alexa fluor 488 conjugated anti rabbit secondary antibody at a dilu tion of 1,100 for 1 hour at room temperature. Sections were mounted in mounting media containing DAPI and visualized with a fluorescence microscope.

RNA extraction and quantitative real time polymerase chain reaction The discs were collected and the AF was separated from the NP. The tissues were stabilized in RNA later in accordance with the instructions of the manufacturer and stored at ?80 C. Total Inhibitors,Modulators,Libraries RNA was extracted with an RNeasy Micro kit in accordance with the instructions of the manufacturer. The DNA template from the RNA preparation was removed by using the DNase I digestion. cDNAs were made by using 0. 5 ug of total RNA, and quantitative Inhibitors,Modulators,Libraries real time reverse transcrip tion polymerase chain reaction was per formed with SYBR Green PCR Master Mix on an ABI 7900 HT sequence detection system. PCR was initiated at 95 C for 10 minutes followed by a 40 cycle amplification phase.

Melting curve analysis was performed to ensure specific PCR product while excluding primer dimers. The data were normalized to the endogenous control, 18S rRNA mes sage, in each sample and were represented as a relative change to the control. All of the primers Inhibitors,Modulators,Libraries used were synthesized by Integrated DNA Technologies, Inc. Sequences for primers are presented in Table 1. Western blot Expression of aggrecan degradation products, MMP 3, MMP 9, and MMP 13 were evaluated by means of Wes tern blot analysis. AF and NP tissue was washed with cold PBS and was extracted with lysis buffer at 4 C for 24 hours. The lysis buffer contains the following, 20 mM Tris HCl, 150 mM Inhibitors,Modulators,Libraries NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2. 5 mM sodium pyrophosphate, 1 mM b glyceropho sphate, 1 mM sodium vanadate, 1 ug mL leu peptin, 1 mM PMSF, and 1X complete protease inhibitors.

The tissue lysate was centrifuged for 10 minutes at 14,000g to collect the clear tissue extract. Protein concentration in the extract was determined by using a Pierce BCA protein assay kit. To detect aggrecan core protein fragments, lysates were treated with chondroitinase ABC 0. 1 U mL in 50 mM Tris acetate EDTA buffer at 37 C for 1 hour. Inhibitors,Modulators,Libraries Protein extracts were centrifuged at 4 C for selleckchem Calcitriol 2 minutes at 14,000g and resolved on NuPAGE 4 12% Bis Tris Gels. Proteins were transferred onto Immo bilon P Membrane.