Detection of Papillomavirus Gene Expression Patterns in Tissue Sections

Heather Griffin1, John Doorbar1

1 Department of Pathology, University of Cambridge, Cambridge
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 14B.7
DOI:  10.1002/cpmc.6
Online Posting Date:  May, 2016
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Abstract

Molecular events during the papillomavirus life cycle can be mapped in infected tissue biopsies using antibodies to viral and cellular gene products, or by in situ hybridization approaches that detect viral DNA or viral transcription products. For proteins, ease of immunodetection depends on antibody specificity and antigen availability. Epitopes in formalin‐fixed paraffin‐embedded (FFPE) samples are often masked by crosslinking and must be exposed for immunodetection. RNA in FFPE material is often degraded, and such tissue must be handled carefully to optimize detection. Viral proteins and viral genomic DNA are both well preserved in routinely processed FFPE samples, with sensitive detection methodologies allowing the simultaneous detection of multiple markers. The combined visualization of nucleic acid and (viral) protein targets, when coupled with image analysis approaches that allow correlation with standard pathology diagnosis, have allowed us to understand the molecular changes required for normal HPV life‐cycle organization as well as deregulation during cancer progression. © 2016 by John Wiley & Sons, Inc.

Keywords: HPV; papillomaviruses; neoplasia; cervix; FFPE; biomarkers; immunofluorescence

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Immunodetection of Viral and Cellular Gene Products In Formalin Fixed Paraffin‐Embedded (FFPE) Tissue
  • Alternate Protocol 1: Signal‐Amplification Systems for the Detection of Low‐Abundance life‐Cycle Markers
  • Basic Protocol 2: Double Staining to Detect Biomarker‐Combinations and to Correlate life‐Cycle Events
  • Basic Protocol 3: Overlaying the Immunofluorescence or Immunohistochemistry Images Onto The H&E IMAGE
  • Basic Protocol 4: Cross‐Reactive Antibodies to the High‐Risk HPV E4 Protein Combined with Mcm Can Confirm the Severity of Cervical Neoplasia
  • Basic Protocol 5: In Situ Detection of HPV Genome Amplification and Co‐Localization of Viral Protein Expression
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Immunodetection of Viral and Cellular Gene Products In Formalin Fixed Paraffin‐Embedded (FFPE) Tissue

  Materials
  • Paraffin‐embedded tissue
  • Xylene, analytical grade (Fisher‐Scientific, cat. no. X/0250/17)
  • Absolute ethanol (Sigma‐Aldrich)
  • Antigen‐retrieval buffer: e.g., 10 mM citrate, pH 6.0 (see recipe) or Buffer D target‐retrieval buffer pH 9.0 (Dako, cat. no. S2368)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Normal goat serum (NGS; Sigma‐Aldrich, cat. no. G9023)
  • Specific primary antibodies against target proteins: e.g., rabbit polyclonal antibody to MCM2 (Abcam, cat. no. ab31159), TVG405 (Doorbar et al., ) directly conjugated to Alexa 488 (Molecular Probes)
  • Fluorophore‐labeled or enzyme‐linked species‐specific secondary antibodies: e.g., anti‐rabbit Alexa 594 (Molecular Probes, cat. no. A11037), anti‐mouse HRP (Southern Biotech, cat. no. 1030‐05)
  • DAPI (4′, 6‐diamino‐2‐phenylindole dihydrochloride; Sigma‐Aldrich, cat. no. D9542), use at 0.5 μg/ml
  • Carrazzi's haematoxylin ×2 (Alpha Laboratories, cat. no. 039‐17705), eosin
  • Superfrost PLUS slides (Thermo Fisher Scientific, cat. no. J1800AMNZ)
  • Coplin jars or staining vessels, and slide holders
  • Hydrophobic pen (e.g., ImmEdge; Vector Laboratories Incorporated, cat. no. H‐4000)
  • Epitope exposure equipment: e.g., microwave, decloaking chamber (Biocare Medical/Menarini Diagnostics), or autoclave, and 1‐L glass or plastic beaker
  • Humidified box
  • Mounting media, e.g., Citifluor (Agar Scientific, cat. no. R1320) or DePeX (VWR, cat. no. 360294H)
  • Coverslips (BDH)
  • Microscopy equipment: brightfield and fluorescent, slide scanner (e.g., Pannoramic Midi Scanner, 3DHISTECH), and software for viewing and acquiring fluorescent images (e.g., Pannoramic Viewer, 3DHISTECH)

Alternate Protocol 1: Signal‐Amplification Systems for the Detection of Low‐Abundance life‐Cycle Markers

  Additional Materials (also see protocol 1)
  • Biotin‐linked species‐specific secondary antibody
  • StrepABC complex (Vector Laboratories)
  • EnVision dextran polymer detection system (Dako)
  • ImmPRESS polymer detection kit (Vector Laboratories), includes 2.5% normal horse serum
  • Tyramide‐fluorophore substrates (fluorescein, tetramethyl‐rhodamine, coumarin, cyanine 3, or cyanine 5; all available from Vector Laboratories)

Basic Protocol 2: Double Staining to Detect Biomarker‐Combinations and to Correlate life‐Cycle Events

  Additional Materials (also see protocol 1)
  • PBS (see recipe)
  • Mouse monoclonal p16 antibody (Santa Cruz Biotechnology, cat. no. sc56330)
  • 0.05% (v/v) Tween 20 in PBS (PBS–0.05% Tween 20)

Basic Protocol 3: Overlaying the Immunofluorescence or Immunohistochemistry Images Onto The H&E IMAGE

  Materials
  • Adobe Photoshop (all recent versions) running on Apple or Windows computer

Basic Protocol 4: Cross‐Reactive Antibodies to the High‐Risk HPV E4 Protein Combined with Mcm Can Confirm the Severity of Cervical Neoplasia

  Materials
  • Formalin‐fixed paraffin‐embedded 5‐μm tissue sections on Superfrost PLUS glass slides
  • PBS (see recipe)
  • 3% hydrogen peroxide in PBS
  • Proteinase K (Qiagen, cat. no. 19131)
  • Digoxygenin (DIG)‐labeled DNA probes, labeled using a DIG DNA labeling kit (e.g., Roche, cat. no. 11175033910)
  • 20× SSC ( appendix 2A)
  • Hybridization buffer (see recipe)
  • Wash buffer (see recipe)
  • Anti‐DIG‐POD, Fab fragments (Roche, cat. no. 11207733910)
  • 1% (v/v) Tween 20 in PBS (PBS–1% Tween 20)
  • Tyramide signal amplification system (Thermo Fisher Scientific, cat. no. T20912)
  • Heat block
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Figures

Videos

Literature Cited

Literature Cited
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