Tunel cell apoptosis kit

We collected granulation tissue samples from both groups on Days 7, 14, and 21 postsurgery to analyze Bcl‐2 and Bax levels using enzyme‐linked immunosorbent assay (ELISA) and to measure granulation tissue apoptosis using the TUNEL assay.
We used Human B lymphocytoma‐2 (Bcl‐2) ELISA kit (enzyme immunoassay, MM‐0381H1) B lymphocytoma‐2 associated X protein (Bax) ELISA kit (enzyme immunoassay, MM‐1143H1), and the TUNEL cell apoptosis kit (Beyotime, C1098).

Cortical tissues were collected, fixed with 4% paraformaldehyde for 24 h and rinsed with running water. Next, the tissues were dehydrated in ascending series of alcohol (70%, 80%, 90%, 95% and 100%) for 30 min, cleared with xylene, embedded in paraffin and cut into 4-μm-thick sections. The sections were stained with TUNEL cell apoptosis kit (C1086, Beyotime Biotechnology Co., Shanghai, China) and apoptotic cells were observed under an inverted fluorescence microscope (HB050; Zeiss, Hamburg, Germany) in six randomly selected visual fields from each section. The percentage of the number of apoptotic cells to the total number of cells was the apoptosis rate.

To calculate the rate of apoptosis in the hippocampal cells of the rats, TUNEL assays were used to detect DNA fragmentation in the nuclei. The rats were anesthetized and transcardially perfused with PBS and 4% paraformaldehyde, after which the entire brain was removed and stored in 4% paraformaldehyde at 4 °C. The brains were washed three times with running water, dehydrated in an alcohol sequence of 70%, 80%, 90%, 95%, and 100% for 30 min, respectively, and embedded in paraffin. The samples were cut into 4-μm sections, which were incubated in PBS containing 0.1% Triton X-100 and 0.1% sodium citrate for 8 min, then washed three times. The sections were washed in 0.3% H₂O₂ in methanol for 10 min and incubated with the reagent from a TUNEL cell apoptosis kit (C1086, Beyotime, China) for 1 h at 37 °C. After imaging under an inverted fluorescence microscope (HB050, Germany), the numbers of apoptotic cells in the hippocampi were calculated in six serial sections using Image-Pro Plus software.