Adiponectin receptor 1

For protein extraction, facet joints were divided into cartilage and cancellous bone and cut into pieces (1 mm³) on silver paper (30 AIS patients and 30 control subjects). The cartilage and cancellous tissues were flash-frozen in liquid nitrogen and homogenized with a traditional mortar or pestle. Then, total protein was extracted with ice cold RIPA Lysis Buffer reagent (CWBIO, China) including PMSF and a phosphatase inhibitor, according to the manufacturer’s instructions. The protein levels were quantified using a bicinchoninic acid (BCA) protein assay reagent (Beyotime, China). Fifteen micrograms of protein for each well were separated by 10% SDS gel and then transferred to a PVDF membrane. The membrane was blocked with defatted milk for 1 h at room temperature, followed by incubation with a primary antibody at 4 °C on a rocker overnight. Primary antibodies included adiponectin receptor 1 (Abcam, USA), RANKL (Abcam, USA), OPG (Abcam, USA), RANK (abcam, USA), adiponectin receptor 1 (Abcam, USA). After incubation with secondary antibodies for 1 h at room temperature, BeyoECL Plus (Beyotime, China) was used to detect the blot. The protein levels were normalized by total protein using TGX FastCast (Bio-rad, USA).

Facet joint tissues were obtained from AIS patients and controls during surgery (22 AIS patients and 15 control subjects). Then, the tissues were formalin fixed, decalcified and paraffin embedded. The paraffin sections were placed in xylene twice and rehydrated with graded alcohol (100%, 95, 85%, 70% alcohol each for 1 min, and double distilled water for 2 min), followed by treatment with 2% hydrogen peroxide for 10 min. After incubation with 5% bovine serum albumin (BSA) for 30 min at room temperature, primary antibodies were added to paraffin sections at 37 °C overnight. Primary antibodies included adiponectin receptor 1 (Abcam, USA) and OCN (Servicebio, China). Then, the sections were incubated with biotinylated goat anti-rabbit immunoglobulins for 30 min, followed by incubation with streptavidin–horseradish peroxidase solution (ZSGB-BIO, China) for 30 min, which bound to the secondary antibody. After washing 3 times with PBS, the sections were stained with 3,3′-diaminobenzidine (DAB) for 30 s. Then, the sections were washed in water, counterstained with hematoxylin (Servicebio, China) for 2 min and observed under the microscope.