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. 2016 Jun 6;117(10):2201–2208. doi: 10.1002/jcb.25606

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© 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.

This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

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Simultaneous detection of two target genes within the same cell using the automated RNA ISH duplex assay. Serial FFPE sections from a head and neck (A, B, E, F, I, J) and cervical (C, D, G, H, K, L) cancer case were stained with the automated RNAscope duplex assay using the Leica BOND RX platform. The automated duplex RNAscope assay was performed to simultaneously detect p16 and the pooled probe HPV‐HR18 (which detects 18 high‐risk genotypes of HPV E6 and E7) (I–L). HPV‐HR18 was detected using DAB (brown) chromogen and p16 was detected using fast red (red) chromogen. (A–H) Sections were probed with either p16 only or HPV‐HR18 only, but detection of both chromogens was performed. Images are shown at either 10× (A, C, E, G, I, K) or 40x (B, D, F, H, J, L) magnification. Black box indicates region shown at 40× magnification.