Immunoguided Laser Assisted Microdissection Techniques for DNA Methylation Analysis of Archival Tissue Specimens Franziska C. Eberle, Jeffrey C. Hanson,

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Immunoguided Laser Assisted Microdissection Techniques for DNA Methylation Analysis of Archival Tissue Specimens Franziska C. Eberle, Jeffrey C. Hanson, J. Keith Killian, Lai Wei, Kris Ylaya, Stephen M. Hewitt, Elaine S. Jaffe, Michael R. Emmert- Buck, Jaime Rodriguez-Canales The Journal of Molecular Diagnostics Volume 12, Issue 4, Pages 394-401 (July 2010) DOI: 10.2353/jmoldx.2010.090200 Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Technical steps for immunohistochemistry of formalin-fixed paraffin-embedded tissue sections on membrane slides for subsequent laser-assisted microdissection and DNA methylation profiling. The Journal of Molecular Diagnostics 2010 12, 394-401DOI: (10.2353/jmoldx.2010.090200) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 CD30 staining of Hodgkin-Reed-Sternberg cells in a Hodgkin's lymphoma on a regular glass slide (A) and on a polyethylene naphthalate membrane slide (B). Both sections of the FFPE sample, either on glass slide or membrane slide, are shown without hematoxylin counterstaining and without cover slide; original magnification, ×40. The Journal of Molecular Diagnostics 2010 12, 394-401DOI: (10.2353/jmoldx.2010.090200) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 CD30+ Hodgkin-Reed-Sternberg cells in a Hodgkin's lymphoma before microdissection (A), selected for microdissection (B), and after microdissection (C) using the Leica LMD 6000; original magnification, ×63. The Journal of Molecular Diagnostics 2010 12, 394-401DOI: (10.2353/jmoldx.2010.090200) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 PCR products after PCR amplification of the β-globin gene using three different sets of primers. A: Detection of 152-bp amplicon. B: Detection of 268-bp amplicon. C: Detection of 676-bp amplicon. P indicates positive control; H, PCR of microdissected tumor cells stained with H&E; M, molecular weight marker; I, PCR of microdissected tumor cells stained with immunohistochemistry; N, negative control. PCR of controls, H, and I were performed as duplicates. The Journal of Molecular Diagnostics 2010 12, 394-401DOI: (10.2353/jmoldx.2010.090200) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 5 Illumina GoldenGate Methylation array of five individual cases. Each plot shows the correlation of target CpG methylation of DNA isolated from H&E-stained tumor cells (x axis) compared with DNA isolated from immunohistochemically stained tumor cells (y axis). A minimum correlation of r = 0.94 and a maximum correlation of r = 0.98 was observed. The Journal of Molecular Diagnostics 2010 12, 394-401DOI: (10.2353/jmoldx.2010.090200) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions