Methyl green

The degenerated IVD was sent for immunohistochemically analysis. Tissue blocks were fixed in 10% formalin, decalcified with 20% EDTA, and embedded in paraffin. Five-micron sections were cut and stained with Safranin-O. Tissue sections were deparaffinized and incubated with proteinase K (25 μg/mL, Sigma) for 60 min at 37 °C. Endogenous peroxidase activity was blocked with 1% hydrogen peroxide. The presence and distribution of Wnt3a and β-catenin was determined using antibodies to detect Wnt3a (1:200 dilution, Santa Cruz Biotechnology) and β-catenin (1:40 dilution, BD Transduction Laboratories) with exposure at 4 °C overnight. Subsequently, a HRP linking 2° Ab was used for 30 min. Bound immunoglobulin was detected using a Dako REAL Envision Detection System (Dako, Carpinteria, CA, USA) and 0.1% methyl green (Dako) was used for counterstaining.

Mandibles from Wdr72^+/+, Wdr72^+/−,Wdr72^−/− litermates (n = 4) were immediately immerse-fixed in 4% paraformaldehyde (PFA)/0.06 M cacodylate buffer (pH 7.3) overnight and decalcified in 8% EDTA (pH 7.2) for 2 weeks at 4 °C. Samples were then dehydrated and paraffin-processed for routine embedding and sectioning. Sagittal incisor sections at 5 μm were deparaffinized and rehydrated, followed by incubation with 10% swine serum for blocking. Rabbit polyclonal antibody to annexin-A8 (Thermo Fisher Scientific) (PA5-31479) was used at 1:500 dilution. Following overnight incubation at 25 °C, sections were washed a biotinylated secondary antibody (Dako, Carpinteria, CA) was added for 1 h at 25 °C. Alkaline phosphatase conjugated to streptavidin (Vector Laboratories Inc., Burlingame, CA) was used to visualize the colorimetric reaction. Sections were then counterstained using methyl green (Dako, Carpinteria, CA).

Mouse mandibles were fixed in 4% paraformaldehyde in 0.06 M sodium cacodylate buffer (pH 7.3) at 4°C for 24 h. After decalcification in 8% EDTA (pH 7.3), samples were processed for routine paraffin embedding and sagittally sectioned. The sections were incubated with 10% swine and 5% goat sera followed by incubation with rabbit anti-human AR (1:75; Novus Biologicals, Littleton, CO, NB100-91658), rabbit anti-mouse TGFBR2 (1:100; Santa Cruz Biotech, Santa Cruz, CA, sc-1700), rabbit anti-human TGFB1 (1:50; Abcam PLC, Cambridge, MA, ab92486), and rabbit anti-mouse PR (Santa Cruz Biotech, sc-166170) antibodies respectively overnight at room temperature. A biotinylated swine anti-rabbit IgG F(ab')2 fraction (Dako, Carpinteria, CA) was used as the secondary antibody for 1 h at room temperature incubation. Following incubation with alkaline phosphatase conjugated streptavidin (Vector Laboratories Inc., Burlingame, CA) for 30 min, immunoreactivity was visualized using a Vector® Red kit (Vector Laboratories) resulting in pink/red color for positive staining. Counter-staining was performed with methyl green (Dako). Negative control was done with normal rabbit sera.