Tunel brightred apoptosis detection kit

To detect cell apoptosis, the cells were seeded on 96-well plates. After transfection, cell apoptosis was detected using a TUNEL BrightRed Apoptosis Detection Kit (Vazyme, Jiangsu, China, A113-01). The TUNEL assay was performed 7 days after transfection in primary SSCs, following the manufacturer’s instructions. In brief, cells were fixed in 4% paraformaldehyde and permeabilized for 10 min using 0.1% Triton-X100 solution at room temperature. Then, the cells were incubated with 50 μl TdT incubation buffer at 37 °C for 1 h in darkness. Finally, cells were stained with DAPI and evaluated under a fluorescent microscope (Olympus, Tokyo, Japan). The ratio of the TUNEL-positive cells (red fluorescence) to the CDH1-positive cells (green fluorescence) indicated the primary SSC apoptosis. Experiments were repeated three times, and each time we randomly selected 5 fields for statistics.

Mouse primary keratinocytes were isolated from newborn fetuses, as described previously 23 (link). The cells were then incubated with primary K15 or K19 antibody. Next, the cells were incubated with secondary antibodies labeled with Alexa Fluor 546 and Hoechst 33342, as described previously 24 (link).
Mouse primary keratinocytes were isolated from Cdc42^{loxp/loxp-Cre-} neonates. The cells were then infected with viruses (vector, plenti-CMV-NLS-Cre (plenti-Cre), 1 × 10⁹ infectious units/ml, purchased from OBiO Technology (Shanghai) Corp., Ltd.); after 3 days, the expression of Cdc42 was detected by Western blotting (WB). Then, the primary keratinocytes were cultured in high-calcium medium (Ca²⁺ concentration increased from 0.09 to 2 mM) for cell experiments (cell-cell junctions and SPRR family members) as described previously 25 (link).
The proliferation of primary keratinocytes was detected with a 5-ethyl-2′-deoxyuridine (EdU) assay (Cell-Light EdU Apollo 567 In Vitro Kit, Ribobio, China). Apoptotic primary keratinocytes were detected using a TUNEL BrightRed Apoptosis Detection Kit (Vazyme Biotech Co., Ltd., China).

Lung sections were prepared as described earlier, and cell apoptosis was detected by TUNEL using the Vazyme^® TUNEL BrightRed Apoptosis Detection kit (A113). TUNEL-positive cells were counted following the manufacturer’s instructions.

The TUNEL assay was applied using TUNEL BrightRed Apoptosis Detection Kit (A113‐03; Vazyme). According to the protocol, cells were fixed with 4% formaldehyde for 25 min and mixed with proteinase K solution for 20 min. Proper concentration of equilibration buffer was added in the samples within time. Then the samples were incubated with reaction mixture for 60 min and cell nuclei were stained with DAPI (C1002; Beyotime). After the samples were washed with PBS, the slides were observed under a fluorescence microscope using × 200 and × 400 times magnification. In order to analyze the data more intuitively, the apoptosis index which means the percentage of positive staining cells in the total number of cells in the microscopic field was calculated in each group.

Cell apoptosis was determined using the TUNEL assay according to the manufacturer’s protocols. The TUNEL BrightRed Apoptosis Detection Kit was obtained from Vazyme (A113, Nanjing, China).

Quantitative analyses for apoptotic cells required TUNEL BrightRed^® Apoptosis Detection Kit (Vazyme, China), following the provided directions. After incubation with the BrightRed Labeling Buffer mixture, whereby wild-type and Golga3^S461L/S461L murine samples were treated to 3 × PBS wash-cycles (5 min each) in the dark,, stained with Hoechst 33342 (5 min, room temperature), mounted with glycerin, and examined and imaged under fluorescence microscopy as above.

Testicular sections (20 µm) from 12-week-old adult WT and KO mice were used for TUNEL staining, according to the manufacturer’s instructions (TUNEL BrightRed Apoptosis Detection Kit, A113-0; Vazyme). The testicular sections were incubated with proteinase K for 20 min at 37°C and then with equilibration buffer (1× TdT) for 30 min at 37°C. Finally, TUNEL reaction mixture and DAPI were used for staining, and the images were visualized with a fluorescence microscope (Axio Imager M2; Zeiss). Random seminiferous tubules (n = 100) were quantified in the TUNEL-positive cells from each testis.