A TdT-mediated dUTP Nick End Labeling (TUNEL) Apoptosis Detection Kit (C1086; Beyotime Biotechnology) was used to detect apoptosis in salivary glands. After paraffin sections were dewaxed with xylene, added proteinase K working solution was added to cover the tissue and incubated at 37 °C for 20 min. The sections were washed three times with PBS (pH 7.4). According to the assay kit, TdT and dUTP were mixed together in a ratio of 1: 9 as the TUNEL reaction solution. The tissue sections were covered with TUNEL reaction solution, placed in a humid box, and incubated at 37 °C for 2 h. After washing thrice with PBS, the sections were stained with DAPI solution (C02-04002, Bioss Antibodies, Beijing, China) in the dark for 10 min at room temperature. Finally, a fluorescence quenching agent (S2110, Solarbio, Beijing, China) was used to seal the sections and evaluate the number of TUNEL-positive apoptotic cells using a fluorescence microscope (Olympus, BX51) at × 200 magnification. The number of green fluorescent cells in these areas were considered to be the number of TUNEL-positive cells.
Dapi solution
Manufactured by Bioss Antibodies
Sourced in China
DAPI solution is a fluorescent staining dye used in molecular biology and microscopy applications. It binds to DNA, allowing for the visualization of cell nuclei. The solution provides a simple and reliable method for nuclear staining.
Automatically generated - may contain errors
3 protocols using dapi solution
1
TUNEL Assay for Apoptosis Detection
2
TUNEL Apoptosis Detection Protocol
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The TUNEL apoptosis detection kit was used (C1089, Beyotime Institute of Biotechnology, Shanghai, China). DNA strand breakage was evaluated by fluorescence labeling of the terminal dUTP notch (TUNEL). DAPI solution (c02-04002, Bioss Biotechnology Co. Ltd., Beijing, China) used nuclear staining. TUNEL positive cells were counted in the defined area and their density was calculated.
3
Quantifying Stress Granule Formation in PC12 Cells
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PC12 cells were grown on glass coverslips in 24 well-plates at 1×105 cells per well. According to the experimental protocol, cells were transfected or co-transfected with miR-335 mimic, miR-335 inhibitor, small interfering (si)-RNA-ROCK2 and pLUC-ROCK2 plasmids, and then stimulated with serum-free medium for 24 h. Following fixation with 4% paraformaldehyde for 30 min at room temperature, PC12 cells were permeabilized with 0.3% Triton X-100 and blocked with 5% BSA. Then cells were incubated with diluted primary antibody (cat. no. 12133-2-AP; rabbit anti-TIA1 antibody; 1:1,000; ProteinTech Group, Inc.) at 4°C overnight. and incubated with the secondary antibody (cat. no. ab150077; Alexa Fluor488-conjugated goat anti-rabbit antibody; 1:5,000; Abcam) for 1 h in the dark. The PC12 cells were rinsed three times with PBS and stained with DAPI solution (BIOSS, Beijing, China) for nuclear labeling. The staining results were observed with a confocal laser scanning microscope (LSM 800; Carl Zeiss AG, Oberkochen, Germany). The formation of SGs was evaluated by calculating the percentage of positive cells with Image Pro Plus software (Version 6.0; Media Cybernetics, Inc.). Each experiment was replicated 3 times and the data are presented as the mean ± SEM.
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