Effects of various decalcification protocols on detection of DNA strand breaks by terminal dUTP nick end labelling.

摘要: To analyse DNA strand breaks by terminal deoxy(d)-UTP nick-end labelling (TUNEL) in calcified tissues including bones and teeth, it is important to decalcify the first. However, effects of decalcifying reagents on integrity are largely unknown. In present study, we evaluated usefulness various 10% EDTA (pH 7.4), 5% trichloroacetic acid (TCA), formic acid, HCl, nitric Plank–Rychlo's solution, Morse's solution K-CX TUNEL staining. Mouse maxilla was selected as experimental system. Apoptotic cells naturally occurring epithelium were analysed. Tissues assessed soft X-ray imaging confirm complete decalcification. The time required for decalcification tissue 7 days with 2 other decalcifiers. Decalcified stained Methyl/Green–Pyronine Y or 4′, 6-diamidino-2-phenylindole assessment integrity. Nuclei epithelial strongly positive both dyes after EDTA, TCA, acid. failed retain DNA. Our results demonstrated good staining treated TCA . Based processing signal-noise ratio, recommend reagent breaks.