Tunel assay kit

For dual immunofluorescence staining, tumor sections were deparaffinized, rehydrated, and the antigen was recovered, permeabilized, and blocked with 5% BSA at room temperature for 1 h. The sections were incubated with primary antibodies at 4 °C overnight, followed by Alexa-Fluor conjugated secondary antibodies (Antgene, ANT023, ANT030) at room temperature for 1 h. Mounting was performed using a fluormount containing DAPI (Antgene, ANT063) for further image acquisition (Olympus BX53).
For TUNEL and immunofluorescence co-staining, tumor sections were deparaffinized, rehydrated, and the antigen was recovered, permeabilized, and blocked with 5% BSA for 1 h at room temperature. The sections were stained using a TUNEL assay kit (Vazyme, A112) according to the manufacturer’s instructions and then incubated with primary antibody at 4 °C overnight, followed by incubation with Alexa-Fluor conjugated secondary antibody (Antgene, ANT029) at room temperature for 1 h. Mounting was performed using a fluormount containing DAPI (Antgene, ANT063) for further image acquisition (Olympus BX53).
Quantification of mean fluorescence intensity and TUNEL-positive proportion was performed using ImageJ software. Five sections were assessed in each group, and four randomly selected viewing fields were evaluated individually in each section.

Haemocytes collected from 30 P. xylostella after different treatments or Drosophila larvae were suspended in 00 μL phosphate buffered saline (PBS, pH 7.4) and plated into the Lab-Tek II Chambered Cover glass (Thermo Fisher, MA, USA) for 30 min at room temperature. Apoptotic haemocytes were visualized using a terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay kit (Vazyme, Nanjing, China) according to guidelines. And 4, 6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) was used for nuclei counterstaining. The images were captured using a confocal microscope, Zeiss LSM 800. To quantify the percentage of apoptotic haemocytes from P. xylostella and D. melanogaster, the labelled nuclei of > 3 images were counted using the Image J software.

The TUNEL assay kit was obtained from Vazyme (Nanjing, China). On the first day, we placed 12-well culture plates in six-well plates and seeded chondrocytes at 80% confluency. On the second day, after the chondrocytes adhered to the plate, we applied the appropriate stimulation to the cells. On the third day, we performed TUNEL staining according to the instructions provided by the kit. Finally, nuclear staining was performed using mounting medium containing DAPI, and the samples were observed under a fluorescence microscope (Nikon ECLIPSE Ti microscope).

The TdT-mediated dUTP nick-end labeling (TUNEL) assay was conducted using a TUNEL assay kit (Vazyme, Nanjing, China) according to the manufacturer’s protocols. The number of TUNEL-positive cells (green) was counted and expressed as a percentage of the green-labeled cells over the total number of ovarian cells (TUNEL index).

Apoptosis was detected using the TUNEL assay kit (Vazyme, Nanjing, Jiangsu, China), according to the manufacturer’s instructions. Paraffin sections were rehydrated and treated with proteinase K, then reacted with TUNEL labeling mix buffer at 37 °C. Images were obtained under a confocal microscope (Zeiss, Oberkochen, Germany). Fifty tubules per male were analyzed.

Cell apoptosis in the liver tissue was detected using a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay kit, according to the manufacturer’s protocol (Vazyme Biotech Co., Ltd, Nanjing, China). After TUNNEL labeling, the liver sections were counterstained with 4′-6-diamidino-2-phenylindole^{40 (link)} to label the nuclei. Images were observed under a fluorescence microscope (Leica Microsystems, Wetzlar, Germany).