Tunel label

To perform the double staining of Anti-FoxP3- (mouse IgG1, monoclonal 236A/E7, 1:50; Thermofisher, Waltham, MA, USA) and TUNEL staining, another protocol was necessary. The procedure resembles immunohistochemical FoxP3 staining, although the endogenous peroxidase was not blocked. After heat pretreatment, unspecific binding sites and staining were blocked by incubation with Ultra-Vision-Protein-Block (Thermofisher, Waltham, MA, USA) for 15 min. The sections were then incubated with the primary antibody FoxP3 for 16 h at 4 °C. After washing with PBS, the secondary antibody goat-anti-mouse-IgG Cy3-labeled (Jackson Immunoresearch Laboratories, West Grove, PA, USA) was applied for 30 min at room temperature. In the next step, the TUNEL staining was performed. TUNEL enzyme (Roche, Basel, Switzerland) and TUNEL label (Roche, Basel, Switzerland) were mixed in a ratio of 1:10 and 50 µL were applied on each slide. Covered with a cover glass, the sections were incubated for one hour at 37 °C. After incubation and washing in PBS, the sections were air-dried and covered with mounting medium for fluorescence with DAPI (Vector, Burlingame, CA, USA).

Cells and tissues were fixed in 2% paraformaldehyde (PFA, Agar Scientific Ltd., Cambridge, UK) for 10 min or 2 h, respectively. Eyes were embedded in optimal cutting temperature compound (OCT, Thermo Fisher Scientific) and cryosectioned (Leica CM1950 cryostat, UK). 14 μm thick cryosections were treated with 0.1% Triton X-100 (Millipore) for 5 min at room temperature followed by 3 washes with PBS. Samples were incubated with TUNEL-MIX, followed by 5% TUNEL-Enzyme in TUNEL Label (Roche). DNase I (50 U/μl, Sigma-Aldrich) treated samples were used as positive control. Negative controls were incubated with TUNEL Label only. The samples were mounted using Vectashield medium with DAPI (Vector Laboratories Ltd., Peterborough, UK).

After treatment with ART for 24 h, cells were fixed in 4% paraformaldehyde at room temperature for 1 h, washed with PBS and permeabilized in 0.1% sodium citrate, containing 0.1% Triton X-100, at 4°C for 20 min. Cells were then resuspended in a final volume of 25 μl of TUNEL reaction mixture (2.5 μl TUNEL-Enzyme in 22.5 μl TUNEL Label, Roche), with the addition of 20 mM EDTA and then counterstained with 5 μg/ml propidium iodide in PBS containing 0.5 μg/ml DNase-free RNaseA, for 1 h at 37°C in a humidified atmosphere in the dark. After being washed with PBS, cells were analyzed by a flow cytometer (Becton–Dickinson, Heidelberg, Germany). Acquisition and analysis of the data was performed using Cell Quest 3.0 software.