Citrisolv hybrid

For immunohistochemistry analysis of human skin tissue samples, formalin-fixed paraffin-embedded sections were deparaffinized with CitriSolv Hybrid (Decon Labs) and rehydrated by sequentially incubating in 100%, 95%, 70%, and 50% ethanol in water. To expose antigenic epitopes, heat-induced antigen retrieval was performed using Antigen Retrieval Reagent-Acidic buffer (pH 6.0) (R&D) for 20min at 95°C. After quenching endogenous peroxidase activity by 3% hydrogen peroxide, sections were incubated with the following primary antibodies at 4 °C overnight (see Supplementary Table 8): NFIB (1:100), NFIX (1:100), K14 (1:2000). After several washes with PBS, primary antibody staining was visualized using peroxidase-conjugated anti-rabbit IgG (1:1000) followed by the DAB substrate kit for peroxidase visualization of secondary antibodies (Vector Laboratories).

CDKN2A FISH was performed only in the two cases with negative p16 IHC. FISH was performed on interphase nuclei using a DNA probe in the short arm of chromosome 9 (LSI CDKN2A localizing to 9p21) labeled in Spectrum Orange simultaneously with a probe for the centromere of chromosome 9 as a control (CEP9 localizing to 9p11-q11) labeled in Spectrum Green (Abbott Molecular, Des Plains, IL). Slides containing FFPE tissue were deparaffinized in Citrisolv Hybrid (Decon Labs, King of Prussia, IL) followed by sequential treatment with 0.2N HCl, 1M sodium thiocyanate, Protease I (Abbott Molecular), 10% formalin, and dehydrating ethanol series (70%, 85%, and 95%). Tissue and probe were codenatured by heating at 80°C for 2 minutes using a ThermoBrite instrument (Abbott Molecular). Hybridization was performed overnight at 37°C in IntelliFISH hybridization buffer (Abbott Molecular). Finally, the slides were mounted with Vectashield containing DAPI (Vector Laboratories, Burlingame, CA). After reviewing the corresponding H&E stained slide with a pathologist the tissue was analyzed by 2 screeners for 200 interphase nuclei per region of interest.