Tmr red tunel cell apoptosis detection kit

The TUNEL assay was extensively carried out for the detection and quantification of apoptosis in histological tissue sections (Duan et al., 2003 (link)). The above tissue sections (n = 3, at each group) underwent staining with TMR (red) TUNEL Cell Apoptosis Detection Kit (Servicebio, G1502, China) as directed by the manufacturer. Following TUNEL labeling, cell nuclei were stained with DAPI (Servicebio G1012, China) for 5 min. Cell apoptosis was assessed by two blinded investigators under a light microscope (Leica, Germany). The nuclei labeled with DAPI appear blue under a fluorescence microscope and the positive apoptotic nuclei were red. The TUNEL-positive cells were recognized as cells double labeled with TUNEL and DAPI and only TUNEL + DAPI were counted. (Li et al., 2018 (link)). Finally, the high power fields of spinal cord were randomly selected, and average percentage in three sections/rat of TUNEL-positive cells to total nuclei were calculated by two blind investigators using ImageJ software (Download from the NIH website²). The apoptotic rate was calculated as AR = (number of TUNEL-positive cells/total number of nuclei) × 100% (Fu et al., 2020 ).

The proliferative ability of primary chondrocytes from WT and Enpp1^−/− mice was detected by EDU staining. Briefly, primary chondrocytes were equably planted in cell culture plates at approximately 50% density, then labeled and stained by using the Click-iT educ-488 cell proliferation assay kit (Servicebio, Wuhan, China).
The 6-μm knee sections (WT and Enpp1^−/− mice) and primary chondrocytes described previously were labeled and stained by TMR (red) TUNEL Cell Apoptosis Detection Kit (Servicebio, Wuhan, China).
The images (EDU and TUNEL staining) were captured by an inverted fluorescence microscope (Leica), and then the proportion of EDU-positive (green fluorescence) and TUNEL-positive (red fluorescence) chondrocytes was analyzed by ImageJ.

The PZT was obtained from SCH Technology Co. Ltd. The 443 nm μ-LEDs were obtained from Shenzhen Rico Photoelectric Technology Co., Ltd. The red μ-LEDs were obtained from Shenzhen Super high-brightness Electronics Co., Ltd. The following reagents and antibodies were used in the study: Anti- IBA 1 Mouse mAb (Servicebio, GB12105; diluted: 1:100), Cy3 conjugated Goat Anti-mouse IgG (H + L) (Servicebio, GB21301; diluted: 1:100), Anti- GFAP Rabbit pAb (Servicebio, GB11096; diluted: 1:100), fluorescein isothiocyanate (FITC)–conjugated goat anti-rabbit IgG secondary antibody (Servicebio, GB22303; diluted: 1:100), TMR (Red) Tunel Cell Apoptosis Detection Kit (Servicebio, G1502-50T; Recombinant TdT enzyme: TMR-5-dUTP Labeling Mix: Equilibration Buffer (1: 5: 50)), Hematoxylin (Servicebio, G1004), Eosin Y (Biobomei, YE2080), 4′,6-diamidino-2-phenylindole (Servicebio, G1012), Toluidine blue (Servicebio, G1032).

5 × 10⁶ cells were subcutaneously injected into four-week-old male BALB/c nude mice. 5 days later, mice were randomly divided into groups and treated with oxaliplatin (5 mg/kg) or 5% glucose solution by intraperitoneal injection, twice per week for three weeks, respectively. All mice were sacrificed and tumors were collected and weighed. Tumor volume equaled length × width² × 0.5 and was measured twice a week. Xenografts were saved in 4% paraformaldehyde for following experiments. Apoptosis rates in the xenograft were determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay using TMR (red) TUNEL Cell Apoptosis Detection Kit (Servicebio, Wuhan, China). Xenograft experiments were approved by the Committee on Animals Handling of Fudan University Shanghai Cancer Center.

TUNEL assay was carried out using the TMR (red) tunel cell apoptosis detection kit (Servicebio, Wuhan, PR China) according to product manual. In brief, paraffin brain sections of tested mice were prepared, deparaffinized, and rehydrated as described in IHC staining. The assay on the prepared sections was carried out according to manufacturer's instruction. In brief, the rehydrated sections were firstly incubated with protease K working solution in PBS at 37°C for 20min. After washing in PBS 3 X 5 min, the sections were permeabilized in PBS containing 0.4% Triton X-100 at RT for 20 min. Then, the sections were washed in PBS 3 X 5 min and equilibrated in equilibration buffer at RT for 10 min. Finally, appropriate volume of tunel reaction solution (TdT enzyme : TMR-5-dUTP labeling mix : Equilibration buffer: 1 : 5 : 50) was applied and the reaction was kept at 37°C for 1 h in a wet box. The following sealing and mounting of the slides for imaging are as described in standard protocol. TMR (Tetramethyl-Rhodamine) glows red by Ex 555/Em 580 nm. The images were captured using Pannoramic 250 Flash III and viewed with CaseViewer 2.4 (3DHISTECH, Budapest, Hungary) and analysed with ImageJ software.

TMR (red)-TUNEL cell apoptosis detection kit (Servicebio, China) was used to investigate whether aconitine induces the apoptosis of neuronal cells in zebrafish larvaes (Quan et al., 2019 (link)). Larvaes at six dpf were treated with aconitine following the methods in Section 2.4. The larvaes were fixed overnight at 4°C in 4% paraformaldehyde (PFA). After washing with PBS, the larvaes were orderly dehydrated by 95%, 90%, 80%, and 70% ethanol for 5 min each and then stored in 100% methanol overnight at -20°C. The larvaes were rehydrated and treated with proteinase K (20 μg/ml) at 37°C for 25 min, and then washed three times with PBS. The samples were added with 0.1% triton to permeabilize the tissues for 20 min at room temperature. The larvaes were then incubated at 37°C in a TUNEL reaction mixture containing TDT enzyme, dUTP, and buffer (1:5:50) for 2 h. After incubation, the nuclei were counterstained with DAPI at room temperature for 10 min. The larvaes were washed with PBS (three times, 5 min each) and covered with an anti-fade mounting medium. Apoptotic cells staining red and nuclei staining blue were observed under a fluorescence microscope.

At the indicated time points, liver tissues were harvested from each group, snap-frozen in liquid nitrogen, embedded in OCT compound, and then cut into 7-μm cryosections. TUNEL assays were performed using TMR (Red) TUNEL Cell Apoptosis Detection Kit (Servicebio, China, G1502) on cryosections under the manufacturer’s instructions. Cell nuclei were counterstained with DAPI (Beyotime, China, C1002). The sections were observed and photographed under a fluorescence microscope (Nikon, Japan). Quantification was conducted with five random fields (200 ×) for each liver tissue section using ImageJ 1.8.0 software (NIH, USA).