Edta antigen retrieval solution

Rat adenohypophysis tissues were fixed in 4% paraformaldehyde (MA0192, Meilunbio, Dalian, China) for 48 h and then made into paraffin sections. After dewaxing and rehydration, the sections were antigenically repaired using EDTA antigen retrieval solution (P0084, Beyotime, Shanghai, China). The samples were sequentially incubated in blocking solution (incubated for 30 min at room temperature), primary antibody (FTO antibody 1:2000 dilution, FOXP2 antibody 1:200 dilution, overnight at 4 °C), and secondary antibody (1:500 dilution, incubated for 1 h at room temperature away from light). The antibodies used were as follows: FTO antibody (ab280081, Abcam, UK); FOXP2 antibody (20,529–1-AP, Proteintech, USA); and anti-rabbit IgG (H + L) antibody (5220–0336, SeraCare, USA). Both antibody incubations were followed by washing three times with PBS (5 min at room temperature). The nucleus was restained with hematoxylin staining solution (C0107, Beyotime, Shanghai, China) after development using a DAB horseradish peroxidase color development kit (P0203, Beyotime, Shanghai, China). The sections were dehydrated and sealed. Images were visualized and collected using an Olympus fluorescence microscope. Three randomly selected images from the immunohistochemistry results were analyzed for positive reaction areas using ImageJ for statistical analysis.

The femora were fixed in 10% neutral-buffered formalin, decalcified with EDTA Decalcified Solution, and embedded in paraffin. Sections (5 μm) were cut along the long axis of samples. Antigen retrieval was conducted by mild heating (100°C) paraffin slides in EDTA Antigen Retrieval Solution (Beyotime, Shanghai, China) for 10 min. Sections were incubated with primary antibodies at 4°C overnight and HRP-linked secondary antibodies at room temperature for 1 h. The primary antibodies were as follows: Cebpb (CST, Danvers, MA, USA), Sp7 (Abcam, Cambridge, UK), and Col2α1 (Santa Cruz Biotechnology, Dallas, TX, USA). The fresh DAB solution was used for chromogenic detection, and hematoxylin was used for nuclear counterstaining. The stained sections were examined and photographed via microscopy at 200x magnification. Immunohistochemical analyses were reviewed and scored by 2 pathologists with over 10 years of experience: 0 (less than 10% cells were stained), 1 (10%-25% cells were stained), 2 (25%-50% cells were stained), 3 (50%-75% cells were stained), and 4 (75%-100% cells were stained).

Paraffin‐embedded TSCC tissues and xenograft tumours were sectioned at 5 μm and mounted onto silane‐coated slides (Thermo Fisher, Waltham, MA, USA), and the sections were de‐waxed in xylene and rehydrated in graded alcohols. H&E staining and IHC were conducted on consecutive tissue sections respectively. IHC was performed according to the manufacturer's protocol (ZhongShan Biotech, Beijing, China). Sections were treated with the EDTA Antigen Retrieval Solution (Beyotime) at 95°C for 15 minutes. The slides were incubated in 3% hydrogen peroxide. Sections were blocked using 5% goat serum and then incubated with primary mouse polyclonal antibody to IRX5 (1:50; Santa Cruz, Texas, CA, USA) and mouse monoclonal antibodies to OPN (1:50; Santa Cruz) and Ki67 (1:100; ZhongShan Biotech) at 4°C overnight. Slides were then incubated with secondary goat anti‐mouse antibody for 15 minutes at 37°C. Diaminobenzidine–HCl (DAB, MXB, Fuzhou, China) was used as a substrate for visualization. Finally, haematoxylin was used to counterstain the nuclei.

For immunofluorescence, cells were seeded on round glass coverslips and subjected to the indicated treatment. Cells were washed with PBS twice and fixed with 4% PFA for 20 min at RT, followed by permeabilization with 0.5% TritonX-100/PBS for 5 min at RT. The cells were then incubated with primary antibodies overnight at 4 °C after being blocked with 3% BSA/PBS for 1 h at RT and followed by incubation with Alexa Fluor-labeled secondary antibodies for 1 h at RT. Nuclei were stained with DAPI.
For tissue immunofluorescence staining, tumors were fixed with 4% paraformaldehyde (PFA) for 4 h at 4 °C. After dehydration and processing, the tumors were embedded in paraffin for sectioning and immunofluorescence. All tissues were sectioned at 6 μm thickness for histological analysis. EDTA Antigen Retrieval Solution (Beyotime, P0085) was used for antigen retrieval. Primary antibodies were incubated overnight at 4 °C after being blocked with 3% BSA/PBS for 1 h at RT. Alexa Fluor-labeled secondary antibodies were used. Nuclei were stained with DAPI.

The fixed tumor tissues were embedded in paraffin and sectioned at a thickness of 5 µm. Thereafter, H&E staining was performed according to the standard procedures. For immunohistochemical analysis, sections were deparaffinized and subjected to antigen retrieval using ethylene diamine tetraacetic acid (EDTA) antigen retrieval solution (Beyotime). The slides were then blocked with 5% bovine serum albumin (BSA) for 1 h and incubated with anti‐CD31 (cat. AF3628, 1:200 dilution, R&D Systems), anti‐Ki67 (cat. 9449, 1:200 dilution, Cell Signaling Technology), and anti‐Cleaved caspase 3 (cat. 9664, 1:200 dilution, Cell Signaling Technology) antibodies overnight at 4 °C. The slides were washed with PBS, incubated with horseradish peroxidase (HRP)‐conjugated secondary antibodies including HRP‐conjugated antimouse (Cat. 7076, 1:400, Cell Signaling Technology), antirabbit (Cat. 7074, 1:400, Cell Signaling Technology), and goat (cat. HAF019, 1:400 dilution, R&D Systems) and then stained with a diaminobenzidine (DAB) kit, followed by counterstaining with hematoxylin. Images were acquired using an Olympus BX 53 microscope and analyzed using Image‐Pro Plus 6.0 software.