Fluoview confocal microscopy system

The TUNEL assay is very sensitive technique to analyze DNA damage in tissue section or cells. In brief, acute leukemia cells were grown in presence or absence of cisplatin, and DNA damage was analyzed by immunochemistry and confocal imaging using Promega DeadEnd™ Fluorometric TUNEL System Technical Bulletin (Cat# G3250) or as previously described [28 (link)]. Both untreated/control and cisplatin treated APL cells were attached on poly-l-lysine coated chambered slide and fixed with 4% paraformaldehyde at 4^°C for 25 min. Fixed cells were permeabilized with 0.2% triton X-100 in PBS for 5 min. Slides were washed with PBS two times and equilibrated in equilibration buffer for 10 min at room temperature. 100 μl rTdT incubation buffer were added on each slide and incubated at 37^°C for one hour. After incubation, the reaction was terminated by dipping slides in 2X SSC for 15 min and washed 2–3 times with PBS. The slides were stained with DAPI and mounted by anti-fade solution with coverslip and nail polish. After drying of slides, confocal imaging was performed using the fluoview confocal microscopy system (Olympus company).

The TUNEL assay is very sensitive technique to analyze DNA damage in tissue sections. In brief, we treated transgenic APL mice with different TX doses (0,1, 2, 4, 6 and 8 mg/kg body wt) and collected the livers in RIPA buffer. Liver tissues were frozen in embedding medium( Polarstat Plus) and 5µM sections were made using Cryostar NX50 (Thermo Scientific, Waltham, MA) and DNA damage was analyzed by immunochemistry and confocal imaging using Promega DeadEnd™ Fluorometric TUNEL System Technical Bulletin (Cat# G3250) or as previously described 33 (link). Liver sections were fixed in acetone and methanol mixture at -20 ⁰ C for 5 min and permeabilized with 0.2% triton X at 4 ⁰ C for 10 min. Slides were washed with PBS two times and equilibrated in equilibration buffer for 10 min at room temperature. 100µL rTdT incubation buffer were added on each slide and incubated at 37 ⁰ C for one hour. After incubation, the reaction was terminated by dipping slides in 2X SSC for 15 min and washed 2-3 times with PBS. The slides were stained with DAPI and mounted by anti-fade solution with coverslip and nail polish. After drying of slides, confocal imaging was performed using the Fluoview confocal microscopy system (Olympus company) 2 (link).