Original ArticlesThe Effect of Aging of Formalin-fixed Paraffin-embedded Tissues on the In Situ Hybridization and Immunohistochemistry Signals in Cervical LesionsNuovo, Allison J. BA*; Garofalo, Michela PhD†; Mikhail, Alexandria‡; Nicol, Alcina F. PhD§; Vianna-Andrade, Cecilia PhD∥; Nuovo, Gerard J. MD†,¶ Author Information *The Ohio State University Medical Center †The Comprehensive Cancer Center, The Ohio State University ‡The Ohio State University, Columbus ¶Phylogeny Inc., Powell, OH §LIPMED-IOC-Fiocruz Institute ∥IFF-Fiocruz Institute, Rio deJaneiro, Brazil Supported by a grant from the Lewis Foundation (to G.J.N.). The authors declare no conflict of interest. Reprints: Gerard J. Nuovo, MD, Phylogeny Inc., 1476 Manning Parkway, Powell, OH 43065 (e-mail: [email protected]). Diagnostic Molecular Pathology 22(3):p 164-173, September 2013. | DOI: 10.1097/PDM.0b013e3182823701 Buy Metrics Abstract Formalin-fixed, paraffin-embedded tissues are widely used in biomedical research but little is known about the effect of the age of the block or unstained slides on the in situ hybridization or immunohistochemistry signal. We compared the in situ-based and immunohistochemistry-based signals for cervical intraepithelial neoplasia samples that ranged from 0 to 15 years of age. There was a progressive and statistically significant decrease in the strength of the p16INK4a signal when comparing tissues prepared from recent unstained slides (0 to 1 y old, mean score of 92%) to those of intermediate age (5 to 7 y old, mean score of 49%) to old unstained slides (cut 13 to 15 y ago, mean score of 10%). Equivalent, progressive, and significant decreases in the intensity of the signals for microRNAs, CD45, and human papillomavirus DNA were seen in tissues stored on slides from 5 to 7 years and 13 to 15 years, respectively. However, the diminution of signal was much less, although still statistically significant, if the sections from the 13- to 15-year-old paraffin blocks were prepared in 2012. The data likely does not represent degradation of the targets as extraction of several microRNA from the old blocks showed no detectable degradation, despite the markedly weakened in situ hybridization signal. It is concluded that in situ-based signal for DNA, microRNAs, and proteins in paraffin-embedded tissues are significantly reduced over time, especially when stored long term on glass slides which, in turn, can lead to a significant underestimation of the amount and presence of the nucleic acid or protein target. © 2013 by Lippincott Williams & Wilkins.