Terminal Transferase dUTP Nick-End Labeling (TUNEL)

Paraffin-embedded eye sections from diabetic and non-diabetic donors were processed with an apoptosis-detection kit (APO-BrdU TUNEL Assay kit; Molecular Probes; Eugene, OR, USA). Samples were deparaffined and rehydrated using standard procedures. To eliminate false positives of TUNEL labeling we pretreated slides with 0.1M citrate buffer for 2 min in a microwave. After rinsing twice with PBS, the sections were incubated with terminal deoxynucleotidyl transferase (TdT) enzyme and deoxythymidine analog 5-bromo-2’-deoxyuridine 5’-triphosphate (BrdUTP) for 90 min at 37 ºC in a humidified atmosphere. At the end of the incubation time, tissue sections were rinsed three times in PBS. Samples were incubated for 1 h in an antibody staining solution containing the Alexa Fluor 488 dye- labeled anti- BrdU antibody. In order to determine the total cellular DNA content, slides were incubated with the Propidium Iodide/ Rnase A staining buffer for 30 minutes. In order to obtain negative control both TdT enzyme and BrdUTP were omitted.

Images were acquired with a confocal laser scanning microscope (FV1000, Olympus. Hamburg, Germany). TUNEL staining in the diabetic retina was compared with that in the non-diabetic retina. For this purpose, each retina was visually scanned with a high power (60x) objective and standardized to a 0.5 cm2 area. The total number of TUNEL positive cells was recorded for each retina in a masked fashion using the valuescorresponding to 15 fields frame images.

Table 1. Clinical features of diabetic and non-diabetic donors included in the study.

Diabetic donors
N= 5 / Non diabetic donors
N= 5 / p
Age (years) / 66.5±8.5 / 64.5 ± 6.4 / n.s
Gender (M/F) / 4/1 / 4/1 / n.s
Diabetes duration (years) / 4.8  5.1
Diabetes treatment
- diet only / 2
- oral agents / 3
- insulin / 0
Cause of death
- Cardiovascular disease / 3 / 2
- Malignant neoplasms / 1 / 2
- Traffic accident / 1 / 1

Figure legend

Figure 3. Cell apoptosis assessed by TUNEL assay in a representative non diabetic retina (A: TUNEL immunofluorescence, C: propidium iodide, E: merged image) and a representative diabetic retina (B: TUNEL immunofluorescence, D:propidium iodide, F: merged image). The bar represents 20 m. G) Quantification of the percentage of apoptotic cells (visually scanned in a masked fashion) in retinal pigment epithelium (RPE), total neuroretina (N), outer nuclear layer (ONL), inner nuclear layer (INL) and ganglion cell layer (GCL) from diabetic (black columns) and non-diabetic retinas (white columns). In all the layers, a high ratio of apoptotic cells were observed in diabetic retinas in comparison with non-diabetic retinas. Results are expressed as the mean  SD. *: p <0.001.