Immunohistochemistry of Intervertebral Disc PAR2

Human cadaveric IVD samples (Table 1) of AF (N = 6), NP (N = 9) and CEP (N = 7) were assessed via IHC using SAM11, a primary antibody for PAR2 (1:50; Invitrogen 35-2300). Briefly, tissue slides were deparaffinized, rehydrated, blocked for endogenous peroxidase activity (0.3% H₂O₂ in MeOH), and antigens retrieved using a citrate buffer (90°C, pH 6.0) for 20 min. Blocking for non-specific binding was performed with 5% goat-serum (1% BSA-PBS, 5% goat serum, 0.05% Tween, and 0.05% sodium azide). Primary antibodies were incubated for 2.5 h in background reducing antibody diluent (Dako S3022) followed by incubation with secondary antibody: biotinylated goat anti-mouse (1:200, VectorLabs BA9200). Finally, tissue sections were incubated with streptavidin-horse radish peroxidase and developed with 3,3-diaminobenzidine (DAB) for 90 s (VectorLabs SK-4100). Tissue was counterstained with Gills No. 2 Hematoxylin (Electron Microscopy Sciences 26030-20) and slides dehydrated and mounted. Normal human lung tissue was used for positive and negative controls, with omission of primary antibody for the latter. All antibody concentrations and DAB times were kept consistent across experimental and control samples. Each tissue sample was quantified as described prior (Wiet et al., 2017 (link)).