TUNEL Staining of Fetal Organ Samples

As previously described [13 (link)], formalin-fixed paraffin embedded fetal organ samples were deparaffinized and rehydrated, followed by protein digestion using Proteinase K solution (15 min). Hydrogen peroxide (3%) was applied (5 min) to block endogenous peroxidase activity, followed immediately by application of the equilibration buffer provided with the ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Millipore Canada Ltd., Etobicoke, Ontario) for 10 s at room temperature. Terminal deoxynucleotidyl transferase (TdT) enzyme was then applied at 37 °C for 1 h in a humidity chamber. Slides were rinsed and the signal was revealed using 3-Amino-9-Ethylcarbazole (AEC) chromogen for 15 min. The whole slides were scanned at 20X magnification using OlyVIA (Olympus Corp., Tokyo, Japan), and converted to JPEG format. Image conversion was conducted using ImageJ (version 1.50i) and the quantitative analysis of TUNEL positive staining was completed using ImagePro Premier software (Media Cybernetics, Inc., Rockville, MD, USA) using the parent-child application for ten random microscopic fields for heart, brain and thymus.