Anti mmp13

Mouse lumbar IVDs were isolated and fixed for two hours at 4°C in 2% paraformaldehyde. The tissues were cryoprotected with 30% sucrose in phosphate buffered saline (PBS, Cat.No.08118006, ThermoFisher Scientific, Pittsburgh, PA) overnight at 4°C, and then embedded in OCT (Cat.No. 4583, Tissue-Tek,Torrance, CA). Serial axial plane lumbar disc cryosections were cut at a thickness of 7 μm. The tissue sections were rehydrated in PBS, permeabilized, and blocked with 0.25% Triton X-100, 10% goat serum and 1% Bovine Serum Albumin (BSA, Cat.No.166099, ThermoFisher Scientific, Pittsburgh, PA) in PBS for 30 minutes at room temperature. Incubation with the specific primary antibodies (anti-Aggrecan, Cat.No. AB1031, Millipore; anti-MMP13, Cat.No. ab39012, Abcam, Cambridge, MA; anti-ADAMTS4, Cat. No. 185722, abcam, Cambridge, MA) were carried out overnight at 4°C following blocking. The sections were then incubated in a secondary antibody Cy3-conjugate goat anti-rabbit IgG (Cat.No.130233, Jackson Laboratory, West Grove, PA) solution for one hour according to the manufacturer’s protocol. Immunostained sections were imaged and analyzed under the Nikon A1 confocal laser microscope and NIS-elements microscopy imaging software.

Proteins were extracted with RIPA buffer containing mixture inhibitors (Roche), followed by homogenization by using QIAshredder (Qiagen) and evaluation of concentration by using a Bradford dye-based assay (Bio-Rad). 50μg of total proteins were loaded on 10% SDS/PAGE and transferred to polyvinylidene difluoride membranes (Hybond; GE Healthcare), followed by immunoblotting with appropriate antibodies. The blots were then incubated with peroxidase linked secondary antibodies followed by enhanced-chemiluminescent detection using ECL kit (Amersham). Primary antibodies were used as followed: anti-HA 1 : 100 (Covance); anti-MMP13 1:500 (Millipore); anti-actin and anti-GAPDH 1 : 10.000 (Sigma-Aldrich).