2.12. IHC analysis

IHC staining was performed to detect collagen type I and II (not pro-collagen type I and II forms) (rabbit, sheep), alginate (rabbit, sheep), GD2 (rabbit), and Tie2 (rabbit). For GD2 and Tie2 staining, NP tissues were embedded in optimum cutting temperature (OCT) compound (Sakura Finetek, Tokyo, Japan) and snap-frozen in liquid nitrogen before fixing them in 4.0% paraformaldehyde (4 °C, 5 min) for sectioning. To prevent NP tissues from spreading into the fixing solution, before sectioning, paraformaldehyde was applied on the whole-frozen (in OCT) samples.

For the rabbit model, mouse monoclonal antibodies to type I collagen (1:100; Sigma-Aldrich, C2456), type II collagen (1:50; Daiichi Fine Chemical, F-57, Tokyo, Japan), or GD2 (1:100; Abcam, ab68456, Cambridge, UK), a goat monoclonal antibody to alginate (1:40 Mochida, CPK-K TB1364), and a goat polyclonal antibody to human Tie2 (1:20; R&D systems, AF313, Minneapolis, MN, USA) were applied. Sections intended for collagen type I and II staining and for alginate staining were deparaffinized in xylene and rehydrated. For IHC of collagen type I and type II, sections were pretreated (15 min) with proteinase K (Dako, Agilent Technologies, Santa Clara, CA, USA), which was not required for IHC to detect alginate. After washing with PBS, sections were treated for 30 min with 1% H₂O₂ in methanol and then incubated with primary antibody for 60 min at room temperature (for type II collagen) or overnight at 4 °C (for type I collagen, alginate, GD2, and Tie2). Sections were then incubated for 30 min in peroxidase (EnVision+ System Kit; Dako) (for type I collagen, type II collagen, alginate, and GD2) or in biotinylated anti-goat IgG + Vectastain ABC kit (Vector USA, Torrance, CA, USA) (for Tie2). Staining was developed using 3,3′-diaminobenzidine hydrochloride (Dako) and Mayer's haematoxylin (Merck, Darmstadt, Germany) as a counterstain. For alginate staining, rabbit NP tissue at 7 days after in vivo UPAL gel implantation was used as a positive control, and NP tissue lacking the primary antibody was used as a negative control. Intact rabbit NP tissue (GD2) and a blood vessel in a rabbit muscle (Tie2) were used as positive controls, and the specimens that were not treated with a primary antibody were used as negative controls (Supplementary Fig. 1).

For fluorescence staining, both anti-GD2 and anti-Tie2 were applied immediately after paraformaldehyde fixation. Then, Alexa-Fluor-568-conjugated donkey anti-mouse-IgG (Invitrogen, Thermo Fisher Scientific, A10037) and FITC-conjugated AffinoPure F-Fragment donkey anti-goat-IgG (Jackson ImmunoResearch, 705–096-147, West Grove, PA, USA) were used as secondary antibodies. Cells positive for type I or type II collagen were separately counted in five independent, randomly selected fields [9]; GD2⁺Tie2⁺ cells were counted in all fields of glass slides. Values are expressed as percentages of positive cells relative to total cell counts. All experiments were performed on eight IVDs from each treatment group and at each time point.

For the sheep model, goat antibody to type I collagen (1:40; Southern Biotech, Birmingham, 1310–01, AL, USA), a mouse monoclonal antibody to type II collagen (1:100; Daiichi Fine Chemical, F-57), and a goat monoclonal antibody to alginate (1:40; Mochida, CPK-K TB136) were used. The sections were deparaffinized in xylene and rehydrated. After washing with PBS, they were treated for 10 min with 3% H₂O₂ in methanol, after which protein blocking was undertaken (Protein Block Serum-Free; Dako). Blocking was not needed for alginate staining. The sections were incubated for 60 min at room temperature with primary antibody and for 30 min with peroxidase (EnVision+ System kit; Dako) (for type I collagen and alginate) or Histofine Simple StainMax-PO (Nichirei Biosciences, Burlingame, CA, USA) (for type II collagen). Staining was developed using 3,3′-diaminobenzidine hydrochloride (Dako) and Mayer's haematoxylin as a counterstain. Cells positive for type I or type II collagen were separately counted, as described above.