Brightred apoptosis detection kit

Apoptosis detection of spermatogenic tubules and cells was conducted using a terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) BrightRed Apoptosis Detection Kit (Vazyme, A113-01), following the manufacturer’s directions. Briefly, the testis sections were equilibrated with TdT buffer for 20 min at room temperature. TdT buffer was removed and terminal transferase reaction mix was added. The reaction was performed for 1 h at 37°C. Sections were washed with PBS and then counterstained with DAPI. Slides were viewed under a LSM700 confocal microscope (Zeiss, Germany).

Apoptosis detection of spermatogenic tubules and cells was achieved using a terminal deoxynulceotidyl transferase nick‐end‐labelling (TUNEL) BrightRed Apoptosis Detection Kit (Vazyme), following the manufacturer's directions. Slides were viewed under an LSM700 confocal microscope (Carl Zeiss AG).

The eye imaginal discs from 3rd instar larvae were dissected in PBS and then fixed in 4% formaldehyde/PBS for 15 min before TUNEL (BrightRed Apoptosis Detection Kit, Vazyme) or immunofluorescence staining. For immunostaining, mouse anti-V5 antibody (1:1000; Invitrogen, Catalogue no. 1461501) or rabbit antiRef(2)P antibody (1:200; Abcam, Catalogue no. ab178440) was used as the primary antibody, and rabbit Alexa Fluor 568 or Alexa Fluor 488 goat anti-mouse IgG (Invitrogen) as the secondary antibody. Tissues were mounted in the anti-fading mounting medium with DAPI (Prolong Antifade; Invitrogen). Fluorescent images were obtained by confocal laser-scanning microscopy (Olympus BX61).

Apoptotic cells were detected by a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL; BrightRed Apoptosis Detection Kit, Vazyme), as previously described (Zhao et al., 2019 (link)). Briefly, BM-MSCs were fixed with 4% (w/v) PFA and treated with proteinase K (10 μg/ml) for 10 min at room temperature, followed by incubation with BrightRed Labeling Buffer for 45 min at 37°C. Cells were washed with PBS three times then stained with DAPI. Images were captured, and TUNEL-positive cells were counted under a confocal laser-scanning microscope (Zeiss LSM800, Carl Zeiss, Oberkochen, Germany).

TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) BrightRed Apoptosis Detection Kit (Vazyme, A113) was used to detect the cell apoptosis according to the manufacturer's instructions. In belief, the embryos and adult tissues sections were hydrated and incubated with protein Kinase (20 μg/ml) at room temperature for 20 min, washed with PBS three times and then incubated with equilibration buffer for 10 min, then TdT (terminal deoxynucleotidyl transferase) reaction mixture was added to the tissues for 1 h incubation at 37°C, washed with PBS three times, incubated with BSA (5 mg/ml) for stopping the reaction if necessary. Sections were counterstained with DAPI for 5 min. The data were obtained using a confocal microscope (Meta Zeiss LSM 780, objective W.I. 63X). If other antibodies were needed to co-incubated at the same section, the sections were blocked by 3% H₂O₂, antigen-retrieval as described before, probed with antibody overnight and then incubated with fluorescence-labeled IgG, all these were operated before protein Kinase incubation.

The apoptotic index of the jejunum was determined using a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) BrightRed Apoptosis Detection Kit (#A113; Vazyme Biotech, Nanjing, Jiangsu, China) in accordance with the manufacturer’s guidance. After deparaffinization and aquation, the jejunal specimens were dipped into proteinase K (20 μg/mL) for 20 min at room temperature, followed by incubation with the TUNEL mixture (deoxynucleotidyl transferase enzyme and BrightRed Labeling Mix) at 37 °C for 1 h in a dark and humidified chamber. Subsequently, the slices were counterstained with the 4,6-diamidino-2-phenylindole solution (#P0131; Beyotime Institute of Biotechnology, Haimen, Jiangsu, China) and the images were captured using a fluorescence microscope (Nikon Eclipse C1; Nikon, Tokyo, Japan). The percentage of TUNEL-positive cells in the jejunum was quantified from 15 well-oriented villi each section by an independent and blinded observer who was not aware of the treatment procedures, using Image Pro Plus 6.0 software.