Gb11022

All the samples were obtained and fixed in 4% paraformaldehyde for 24 h. They were later embedded in paraffin. Histology slices were made on a rocking microtome. Hematoxylin and eosin staining was performed to observe the pathological dynamics in masses. To detect the cell proliferation in the tumor tissue, the sections were incubated with the Ki-67 antibody (1:500, GB13030-2, Servicebio, China) and detected using goat anti-rabbit IgG H&L (HRP) antibody (1:200, GB23303, Servicebio, China). DAB (DAKO, K5007, Japan) was used as the chromogen and then the section was counterstained with hematoxylin. The positive percentage calculated by four pathological professionals without prior knowledge of the experimental process. To detect the fibroblasts infiltration and blood vessels in the tumor tissue, the sections were incubated with collagen 1A1 (1:1,000, GB11022, Servicebio, China) and α-SMA (1:500, GB111364, Servicebio, China) antibody. The following procedure was the same with Ki67 staining. To detect the cell origin in the tumor, the sections were stained with anti-HLA G antibody (1:200, ab7758, Abcam) and detected with goat anti-mouse IgG H&L Alexa Fluor 488 (ab150113).

Aortas fixed in formalin and embedded in paraffin were sectioned at 5 μm. Masson or wheat germ agglutinin (WGA) staining was performed using standard procedures. For immunofluorescence staining, the paraffin-embedded and frozen sections of the heart were incubated with primary antibodies for α-SMA (1:100) (19245S, CST, Danvers, MA, USA), Col1a1 (1:100) (GB11022, Servicebio, Wuhan, China), and F4/80 (1:100) (ab6640, Abcam, Cambridge, UK). Antigen retrieval of the paraffin section was obtained by heating the tissue slides in 0.01 M citrate buffer, pH 6.0, at 100°C for 5 min.

To compare cartilage extracellular matrix (ECM) expression levels of chondrocytes at P1 and P3, cell-seeded coverslips of AUCs were firstly prepared. Briefly, after coverslips (24-mm diameter) were placed in a six-well plate, chondrocyte suspensions of P1 or P3 were added, and plates were cultured in an incubator with 5% CO₂ at 37°C. After culture for 48 h, immunofluorescence staining of type II collagen (COL II), type I collagen (COL I), and type X collagen (COL X) was performed using rabbit polyclonal antibody (1:100 in PBS; ab34712, Abcam, Cambridge, United Kingdom), rabbit polyclonal antibody (1:500 in PBS; GB11022, Servicebio, China), and rabbit polyclonal antibody (1:200 in PBS; DF13214, Affinity Biosciences, United States), followed by incubation with Cy™3 AffiniPure Goat Anti-Rabbit IgG (H + L) (red, Jackson ImmunoResearch, West Grove, PA, United States). Alcian blue staining was performed to detect expression of glycosaminoglycans (GAG) following standard histological protocols (Cooke et al., 2018 (link)).

Our previous study also described this method in detailly [2 (link)]. After intervertebral disc sections or NP cells were well prepared, 0.1% Triton X-100 was employed to permeate samples for 5 min. Then, samples were blocked with 3-5% BSA for 60 min at 37°C. Then, the samples were incubated using primary antibody against γH2AX (GB111841, 1 : 200, Servicebio, Wuhan, China), aggrecan (GB11373, 1 : 500, Servicebio, Wuhan, China), collagen type II (GB14073, 1 : 500, Servicebio, Wuhan, China), iNOS (GB11119, 1 : 1000, Servicebio, Wuhan, China), collagen type I (GB11022, 1 : 500, Servicebio, Wuhan, China), NLRP3 (GB11300, 1 : 1000, Servicebio, Wuhan, China), CD206 (#24595, 1 : 800, Cell Signaling Technology, Inc. USA), MMP3 (GB11131, 1 : 500, Servicebio, Wuhan, China), AGT (ab108334, 1 : 200, Abcam, USA), Nrf2 (340675, 1 : 500, Zenbio, Chengdu, China), and p65 (GB11142, 1 : 200, Servicebio, Wuhan, China) at 4°C overnight. At the second day, samples were treated with fluorescence secondary antibody (GB22303, GB21301, Servicebio, Wuhan, China) for 1 h in the dark room. The nuclei were staining with DAPI solution (G1012-100ML, Servicebio, Wuhan, China). The fluorescence was detected using fluorescence microscope (Olympus, Japan).

Histological and immunohistochemical analyses were performed to evaluate the regenerated bone in the samples using hematoxylin and eosin, safranin‐O/fast green, and Masson's staining. Immunohistochemical staining of COL2 and OCN was also performed to assess the histological structure and special matrix deposition, respectively. The COL2 and OCN expressions were detected using rabbit polyclonal antibodies against COL2 (ab34712, Abcam, Cambridge, USA) and OCN (GB11022, 1: 500, Servicebio, Wuhan, China), followed by 3,3′‐diaminobenzidine tetrahydrochloride solution. The sections were counterstained with hematoxylin to produce a brown precipitate at the antigenic site. The stained slides were examined and photographed using a microscope (Pannoramic MIDI, 3D HISTECH).

Tissue sections were prepared at a thickness of 4 μm and stained with hematoxylin, eosin, and Masson according to standard procedures and liver histology was assessed using an Olympus BX53 + DP80 upright fluorescence microscope imaging system (Olympus, Tokyo, Japan). According to the microscopy, three sections were chosen from each group for IHC analysis. Briefly, the sections were dewaxed, washed with water, placed in citric acid antigen repair buffer (pH 6.0) for antigen repair, and washed with phosphate-buffered saline (PBS). The sections were placed in 3% hydrogen peroxide solution, incubated at room temperature in the dark for 25 min, and washed with PBS. Then, 3% bovine serum albumin (BSA) was added to block at room temperature for 30 min. The slides were treated with primary antibodies against α-SMA (1:2000, Servicebio, Wuhan, China, GB111364) and COL-1 (1:1000, Servicebio, Wuhan, China, GB11022) overnight at 4 °C and then incubated with the appropriate secondary antibody (1:200, Servicebio, Wuhan, China, GB23303) (HRP-labeled goat anti-rabbit). Reaction products were visualized using diaminobenzidine (DAB, Servicebio, Wuhan, China) and monitored by microscopy.