Nanozoomer s210 digital slide scanner

L6/S1 motion segments were fixed in zinc formaldehyde and histological staining including IHC was performed as described below. Following fixation, IVD samples were decalcified with 23% formic acid solution with three changes over 72 h. After decalcification, samples were dehydrated with 2-etoxyethanol and cleared with benzyl benzoate. Post dehydration and clearing, samples were infiltrated and embedded with a custom hydrophobic resin. Embedded samples were trimmed, and 5-μm thick sections were cut with a tungsten carbide knife and mounted on adhesive microscope slides. Thin sections were plasticized using xylene and stained with Alcian Blue (AB) and Picrosirius Red (PSR) [35, 36] . Briefly, slides were stained with Weigert's hematoxylin for 10 min before staining with AB solution for 30 min and PSR for 1 h as described [35] . Slides were imaged at 20X on the Hamamatsu NanoZoomer S210 Digital Slide scanner and degenerated grades scored via standardized grading system [37] by 3 separate graders and averaged. This comprehensive scoring system accounted for the entire IVD regions of NP (cellularity, morphology, fibrosis, matrix organization), AF (cellularity, bulging, lamellar organization, clefts/fissures), the cartilage endplates (cellularity, fissures/microfractures, Schmorl's nodes), and interfaces of these different regions.

For FOXF1, thin sections were deplasticized using xylene for iIHC. FOXF1 (GeneTex GTX48981) rabbit polyclonal antibody was used at a dilution of 1:500 and detected with a polymer, horseradish peroxidase linked anti-rabbit secondary. DAB (diaminobenzadine) was used as the visualization chromogen. Images were then scanned using the Hamamatsu NanoZoomer S210 Digital Slide scanner and quantified by % positive cells over total cells in each NP, AF, and CEP region.