Converter pod

For the apoptosis analysis, LM3 cells were treated with capsaicin, sorafenib or their combination for the indicated times, trypsinized and collected in tubes, and incubated in binding buffer with propidium iodide and FITC-conjugated Annexin V for 10 min in the dark at room temperature. Flow cytometry analysis was used to estimate the cell apoptosis rate. The TUNEL method was applied to visualize the 3′-OH ends of DNA fragments in apoptotic cells according to the manufacturer's protocol (Roche Diagnostics GmbH). LM3 cells were subjected to different treatments for 48 h and fixed in 4% paraformaldehyde. The cells were then submerged in methanol containing 0.3% H₂O₂ to inhibit endogenous peroxidase activity. Following washing with PBS, cells were covered with proteinase K solution for 10 min. Subsequently, the cells were covered with the TUNEL reaction mixture and incubated for 1 h at 37°C in the dark. After washing in PBS, the cells were placed in Converter-POD (Roche Diagnostics GmbH) and then incubated at 37°C for 30 min. After rinsing in PBS three times for 5 min, the cells were dipped in DAB (Roche Diagnostics GmbH) at room temperature for 10 min and observed under a microscope.

The paraffin-embedded sections (5-µm thick) of retinal tissues were deparaffinized, treated with 0.1% Triton X-100 and blocked with 3% H₂O₂. After washing with PBS, the sections were incubated with the mixture of terminal deoxynucleotidyl transferase (TdT) and fluorescein-labeled dUTP and then treated with converter-POD according to the manufacturer's protocol (Roche, Basel, Switzerland). DAB was added and the sections were counterstained with hematoxylin. Finally, the sections were dehydrated and photographed under OLYMPUS DP73 microscope.

TUNEL staining was used to measure the rate of apoptosis, as previously described (16 (link)). Following 3 h reperfusion, rat hearts were removed. Myocardial tissue sections were dehydrated in ascending series of ethanol, cleared in xylene and embedded in paraffin. Paraffin-embedded myocardial tissues were cut into sections 5-µm thick, dewaxed with xylene and stained for TUNEL analysis. Sections were penetrated for 8 min with 0.1% Triton X-100 (50 µl; Beyotime Institute of Biotechnology, Haimen, China) and washed 3 times for 5 min each in PBS at 37°C. Sections were then blocked for 10 min by 3% H₂O₂ and washed 3 times for 5 min each in PBS at 37°C. TUNEL reaction mixture was added to the sections, which were then incubated for 60 min at 37°C in a dark in a humidified environment. Sections were washed 3 times in PBS for 5 min each at 37°C. Converter-POD (50 µl; Roche Diagnostics, Basel, Switzerland) was added to the sections and incubated for 30 min in a humid environment at 37°C. Sections were washed 3 times for 5 min each in PBS at 37°C. Sections were incubated with DAB substrate for 10 min at 15°C and washed 3 times for 5 min each in PBS at 37°C. Finally, sections were stained by hematoxylin for 3 min at 37°C and fixed by neutral balata. TUNEL-positive cells were counted using a fluorescent microscope (Olympus, Tokyo, Japan) at a magnification of ×400.

For TUNEL staining, an in situ cell death detection kit with horseradish peroxidase (POD; Roche, Basel, Switzerland) was used according to the manufacturer’s protocol. After deparaffinization and rehydration, sections were incubated with protease K (40 μg/mL) for 15 min at 37°C and 3.0% hydrogen peroxide for 5 min to remove endogenous peroxidase. The samples were immersed in TUNEL reaction mixture for 75 min at 37°C in the dark, followed by incubation with Converter-POD (Roche) for 30 min. Images were captured by optical microscopy.