Tunel staining

Paraffin embedded sections of human and murine tissues were subjected to haematoxylin and eosin (H&E) or immunofluorescence staining with antibodies directed against keratin 5 (rabbit, Abcam), CADM1 (chicken, MBL), E-cadherin (HECD1, mouse, Abcam), BrdU (rat, Abcam), Luciferase (rabbit, Abcam), keratin 14 (rabbit, Covance), and phospho-STAT3 (Cell Signalling). Detection of apoptotic cells was via TUNEL staining (Promega). Immunostained cells were fixed using 4% paraformaldehyde and incubated with antibodies to CADM1 (chicken, MBL), ITGA6 (rabbit, R&D), and HER2 (Rabbit, Cell Signalling).

Tumor tissues from vehicle and 150 mg/kg YL529 groups were fixed in 4 % paraformaldehyde, dehydrated and embedded in paraffin [27 (link)]. Sections of these tissues were subsequently incubated with LYVE-1 and corresponding second antibodies and visualized using peroxidase-DAB. TUNEL staining was also performed for fixed tissues (Promega, USA). Quantification was done as described [28 (link)]. Briefly, the microvessel counting was done in representative 200× fields or three high-power (400×) fields of the highest vascular density.

The donors agreed to use human-derived cartilage samples, and this study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Guangxi Medical University (Approval number: 2022-E320-01). Cartilage sample sections were pretreated with 3% hydrogen peroxide, blocked with 10% goat serum (Gibco), and probed with PDK1 (1:400) primary antibody overnight at 4°C. Unbound antibodies were washed away with PBS and incubated with IgG secondary antibody (1:300, Thermo Fisher Scientific) for 2 h at room temperature. Next, diaminobenzidine (DAB) substrates (Boster, China) were used to develop the positively stained areas, and sections were counterstained with hematoxylin. Tunel staining (Promega) was performed to determine the level of apoptosis in sectioned bone tissues. Finally, sections were observed using an upright microscope (Olympus).

Four- to six-week-old female nude (BALB/C nu/nu) mice, purchased from Sun Yat-Sen University’s Laboratory Animal Canter (Guangzhou, China), were used to generate a subcutaneous xenograft mouse model. SW480 cells transduced with lenti-NRBP1 or lenti-GFP were harvested, and approximately 1 × 10⁷ cells in 200 μL PBS were injected subcutaneously into each mouse. Mice were monitored daily, and tumour growth was evaluated every five days by measuring the length and width of the tumour mass with callipers. The tumour volume was calculated by the following formula: (length × width²)/2. Twenty-five days after injection, the mice were killed, and the tumours were removed and weighed. The tumour tissues were fixed in 4% formalin and then embedded in paraffin for further IHC staining and TUNEL staining. TUNEL staining was conducted following the manufacturer’s instructions (Promega, Madison, WI, US). All animal procedures were conducted following the Guide for the Care and Use of Laboratory Animals and were approved by the Animal Experiment Administration Committee of the Medical School of Sun Yat-sen University.

For the analysis of brain histopathology 15-week-old WT and Zfp106^−/− mice were perfused transcardially with saline followed with 4% PFA, brain transferred to formalin and embedded in paraffin wax. Staining with H&E and anti- GFAP, IBA-1, p62, MBP and neurofilament antibodies was undertaken using a Ventana automated immunohistochemical staining machine (Ventana Medical Systems, Tuscon, AZ, USA). Paraffin-embedded sections once incubated with appropriate secondary antibodies were developed using 3,30-diaminobenzidine and counterstained with haematoxylin.
Twenty micrometer transverse sections from the lumbar region of fixed spinal cords collected onto glass slides were immuno-fluorescently stained with anti-GFAP (Cy-conjugated mouse monoclonal, Sigma; used at 1:1000) and anti-IBA1 (rabbit polyclonal, Abcam; used at 1:500), visualized using a secondary AlexaFluor488-conjugated antibody (Invitrogen) and counterstained with NeuroTrace® 435⁄455 fluorescent Nissl stain (Invitrogen). A C-terminal p62 antibody (Progen, Germany; 1:300) was also used to stain lumbar spinal cord sections. Confocal images were taken using a Zeiss 710 microscope. Active cell death was analysed using TUNEL staining (Promega) following manufacturer recommendations.

The cardiomyocytes (5 × 10⁵ /well) were seeded in 6‐well plates. After H/R treatment, the cardiomyocytes were washed twice with PBS and fixed with 4% paraformaldehyde. TUNEL staining (Promega) was used to visualize apoptotic cells following the manufacturer's instructions. The percentage of positive cells was calculated by TUNEL fluorescence density/DAPI fluorescence density, and the density was analysed using ImageJ software 1.26.

Apoptotic cell death in kidney sections was determined using TUNEL staining (Promega, Madison, WI, USA). Briefly, kidney sections were deparaffinized and pretreated with 0.1 M sodium citrate, pH 6.0, at 65 °C for 30 min and then incubated with a TUNEL reaction mixture for 1 h at 37 °C in a dark chamber, and the nuclei were labeled by DAPI. Positive staining with nuclear DNA fragmentation was detected by fluorescence microscopy (Zeiss, Germany).