Collagen Type II Immunofluorescence Staining in IVD

Collagen type II (Col II) distribution in the IVD was analysed by immunofluorescence staining. Antigen retrieval was performed in paraffin sections through incubation with 20 μg/mL proteinase K (Sigma-Aldrich Inc., St Louis, MO, USA) solution for 15 min at 37 °C. Sections were incubated overnight at 4 °C with the anti-collagen II-II6B3 (Developmental Studies Hybridoma Bank, 1:20 dilution) antibody followed by incubation with Alexa Fluor 594-labeled goat anti-mouse antibody (1:1000, Molecular Probes, Invitrogen, Carlsbad, CA, USA). Sections were mounted in Fluorshield with DAPI (Sigma-Aldrich Inc., St Louis, MO, USA). Representative images of the slides were collected using an inverted fluorescence microscope (Axiovert 200 M, Zeiss, Jena, Germany). The %COL2 in the NP was determined as previously described [20 (link)].