Tunel staining kit

The death of neurons in injured spinal cords was detected by a TUNEL Staining Kit (Servicebio, Wuhan, China). Briefly, after treating with 0.1% TritonX-100 (Biosharp) for 20 min, the paraffin slices of spinal cords were incubated with the mixture including terminal deoxynucleotidyl transferase (TDT, Servicebio) enzyme, deoxyuridine triphosphates (dUTP, Servicebio), and buffer at a ratio of 1:5:50 at 37°C for 2 h. The sections were counterstained with DAPI and observed under a fluorescence microscope (Leica).

The renal tissues were preserved in 4% paraformaldehyde, embedded in paraffin blocks, and cut into 10 μm sections. To investigate apoptosis, terminal deoxynucleotidyl transferase d-UTP nick end labeling (TUNEL) assay was performed by using a TUNEL staining kit (Servicebio, Wuhan, China) in adherence to the manufacturer's instructions. After treatment with the assay kit, the sections were observed under a fluorescence microscope and images were collected for quantification using Image J. This kit was labeled with CY3 fluorescein and the positive apoptotic nuclei were red.

The femoral head samples were fixed using 4% paraformaldehyde, decalcified with 10% EDTA solution, embedded in paraffin, and then cut into 5 μm sections with a microtome. The sections were deparaffinized with xylene and rehydrated in a graded series of ethanol solutions, before being stained with hematoxylin and eosin (H&E). H&E staining was performed to detect pathological characteristics and changes in the femoral heads from different groups, and the slices were observed under a light microscope (Nikon Ni-U, Japan).
TRAP staining was used to identify osteoclast cells of each group. The slides were incubated with TRAP staining solution (Servicebio, Wuhan, China) for one hour in the dark. The osteoclasts were defined as cells that are TRAP-positive with more than three nuclei. The apoptotic cells were detected with a TUNEL staining kit (Servicebio, Wuhan, China). Apoptotic cells appeared brown.

The tumor tissues specimens of mice were fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. Then the tissue blocks were cut into sections of 3-5 um thickness. For Ki67 staining, the sections were immunostained overnight at 4°C with an anti-Ki67 antibody (Abcam). After washing in PBS, the sections were subsequently incubated with the second antibody for 1 h. Then the slides were stained with 3, 3-diaminobenzidine (DAB) and hematoxylin separately, dehydrated, and mounted. Images were captured using a microscope (Leica, Leica DMi8). For TUNEL staining, the proportion of apoptotic cells were performed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining kit (Servicebio, Wuhan, China). We obtained all images using a CLSM.

After exposed to 2 × 10⁻⁴ m H₂O₂ in serum‐free α‐MEM for 4 h, the senescence of TDSCs was investigated by SAβ‐gal staining and γ‐H2AX immunofluorescence staining and following manufacturer instructions (Cell Signaling Technology, USA).^[62] TUNEL staining kit (Servicebio, China) was used to detect cell apoptosis according to instructions. Briefly, after the cells were fixed and permeabilized with Proteinase K, TUNEL reaction buffer was added for reaction for 60 min. Subsequently, the slides were observed and recorded using DMi8 fluorescent microscopy. The apoptotic status of cells was analyzed by flow cytometer and visualization of flow cytometry data was performed using FlowJo 10.9.1 software. Briefly, after coincubation with H₂O₂, TDSCs were washed with PBS gently, and then followed by incubation with Annexin V‐FITC and propidium iodide (PI) staining solution for 20 min away from light. Then the samples were analyzed by flow cytometer (CytoFLEX, USA) immediately, and data were analyzed by FlowJo software.

Apoptosis of intestinal tissue was determined using the TUNEL staining kit (G1501, Servicebio) according to the manufacturer’s instructions. All samples were then photographed and examined under a fluorescence microscope.

The DRG tissue (L4–L6) was embedded in paraffin with a thickness of 5 microns. After dewaxing hydration, repairing with protease K and breaking with 0.1% triton, the TUNEL staining kit (Servicebio; G1501) was used for coloring, and the cell nuclei were re-dyed with DAPI. The samples were sealed with anti-fluorescence quenching sealing reagent, and the images were observed and collected under a fluorescence microscope.