Immunohistochemical Analysis of Facet Joint

IHC procedures were previously reported.²⁶ Facet joint tissues were fixed with 10% formalin for 3 days, decalcified by 10% formic acid for one week and embedded in paraffin. The paraffin blocks were cut into 5–7 mm thick sections. Briefly, tissue slides were deparaffinized, dehydrated, incubated with Antigen Retrieval Kit (VitroVivo Biotech, VB‐6009), permeabilized with 0.3% Triton X‐100 in 1× TBS‐T and eliminated endogenous peroxidase with BLOXALL (Vector Lab, SP‐6000). Tissue slides were then followed by incubation for overnight at 4°C with the appropriate primary antibodies in antibody diluent (DAKO, S3022), ABC kit components (Vector Lab, PK‐6102), DAB substrate kit (Vector Lab, sk4100), counterstaining with haematoxylin (Merck, 1.05174.0500) and mounting with Permanent mounting medium (Vector Lab, H‐5000). Images were collected with a Nikon eclipse Ti‐U microscope. Five fields for each sample were acquired randomly at 200× magnification.