Histological and Immunohistochemical Evaluation of IVD Degeneration

After 4% paraformaldehyde fixing for 48 hours, IVD tissue samples were decalcification in the 10% EDTA for 21 days and then embedded into paraffin blocks. After being subjected to 5 μm thickness histological sectioning, hematoxylin and eosin (H&E) staining and safranin O-fast green staining were then processed for histological assessment under standard laboratory protocols. All sections were captured using the high-quality microscope (Leica DM4000B). The histological score was evaluated based on the standard evaluation of 5 categories of degenerative changes [27 (link)]. For immunohistochemical evaluation, tissue sections were first dewaxed by graded xylene and standard alcohol gradients. After washing with PBS and water, 10% goat serum was used to block for 30 min at RT. Subsequently, the sections were incubated with the primary antibodies (anti–MMP9 and anti–MMP13 purchased from Servicebio, Wuhan, China) overnight at 4°C. The next day, the appropriate horseradish peroxidase labeled secondary antibody was incubated with the sections for 1 hour at RT and then developed with diaminobenzidine solution. Finally, all sections were captured under a high-quality microscope (Leica DM4000B).