Immunohistochemistry (IHC)

Immunohistochemical analysis was performed in serial sections for ECM proteins (Aggrecan, Collagen 2), the notochordal cell marker (Brachyury), stem cell marker (Oct4), chondrocyte marker (Sox 9) and Green Fluorescent Protein (GFP) using the Vectastain^tm rabbit or mouse kit (Vector Labs, ON). Briefly, after Safranin-O staining, subsequent serial tissue sections were deparaffinized in xylene followed by hydration in gradient alcohol. Antigen retrieval was performed using microwave-based heat retrieval method. Thereafter, slides were washed three times with Tris-buffered saline (TBS, 1X, pH = 7.4) containing 0.025% Triton-X-100 (TBS-T), followed by blocking with appropriate serum, provided in the kit. The slides were then incubated with rabbit polyclonal or mouse monoclonal primary antibodies at appropriate dilutions for 1 h at room temperature followed by washing with TBS-T, three times). The sections were incubated with hydrogen peroxide (0.3% v/v) for 15 min to block endogenous peroxidase activity, followed by three washes with TBS-T. Tissue sections were incubated with the goat anti-rabbit or mouse secondary antibody at appropriate dilution for 30 min. Protein expression was detected using diaminobenzidine (DAB) as chromogen. The sections were counterstained with Meyer's hematoxylin and mounted with DPX mountant. For scoring, ECM and cytoplasmic staining for Aggrecan, Col2A1 and nuclear expression of Brachyury, Oct4 and Sox-9 was considered as positive staining for IHC analysis (Supplementary Table ^S4). The bright field sections were evaluated semi-quantitatively for % positivity and staining intensity in IVD-NP tissue by light microscopic examination using a Nikon bright field microscope (Nikon Eclipse TE2000-U).