Detection of Cell Proliferation Markers by Immunofluorescence Staining and Microscopy Imaging in Paraffin‐Embedded Tissue Sections

Seda Eminaga1, Polakit Teekakirikul2, Christine E. Seidman3, Jonathan G. Seidman3

1 Cardiovascular Division, King's College London, London, 2 Heart and Vascular Institute, Pittsburgh, UPMC Presbyterian, 3 Department of Genetics, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 14.25
DOI:  10.1002/cpmb.13
Online Posting Date:  July, 2016
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This unit describes a step‐by‐step protocol to detect and quantify proliferating cells in paraffin‐embedded tissue sections. Two well‐established markers of proliferation (incorporation of BrdU into newly synthesized DNA and expression of the nuclear protein Ki67) are detected after antigen‐retrieval and subsequent immunofluorescence staining and confocal microscopy. © 2016 by John Wiley & Sons, Inc.

Keywords: BrdU; cell proliferation; immunofluorescence; immunohistochemistry; Ki67

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Immunofluorescent Detection of BrdU or Ki67 in Paraffin‐Embedded Tissue Sections
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Immunofluorescent Detection of BrdU or Ki67 in Paraffin‐Embedded Tissue Sections

  • 5‐μm thick paraffin‐embedded tissue sections mounted on positively charged glass slides (Superfrost Plus, Thermo Scientific, cat. no. J1800AMNZ)
  • Xylene (histological grade, Sigma)
  • 70%, 80%, 95%, and 100% (v/v) ethanol (histological grade, Sigma)
  • Phosphate‐buffered saline (PBS; appendix 22)
  • Tween‐20 (Sigma, cat. no. P9416)
  • Antigen retrieval buffer (see recipe)
  • Tris‐buffered saline (TBS, see recipe)
  • 100,000 U/ml exonuclease III (Exo III, New England Biolabs, cat. no. M0206S)
  • 20,000 U/ml EcoRI and 10× EcoRI buffer (New England Biolabs, cat. no. R0101S)
  • TBS with 0.1% (v/v) Tween‐20 (TBS‐T)
  • Blocking buffer: TBS containing 10% normal goat serum (Vector Laboratories, cat. no. S‐1000)
  • Antibody diluent (Dako, cat. no. S0809)
  • Primary monoclonal antibody:
  • Mouse anti‐BrdU clone Bu20a (Dako, cat. no. M0744)
  • Rat anti‐BrdU clone BU1/75 (ICR1) (Abcam, cat. no. ab6326)
  • Rat anti‐Ki67 clone SolA15 (eBioscience, cat. no. 14‐5698‐82)
  • Species‐matched IgG: mouse IgG1 (Dako, cat. no. X0931) or rat IgG (Vector Laboratories, cat. no. I‐4000)
  • Alexa Fluor 488–conjugated goat anti‐mouse or anti‐rat secondary antibody (Life Technologies, cat. nos. A11001 or A11006)
  • Mounting reagent containing DAPI (Prolong Gold Antifade Mountant with DAPI, Life Technologies, cat. no. P36935)
  • Staining racks and vessels with lids (xylene‐ and heat‐resistant; Tissue‐Tek Slide Staining Set, Sakura Finetek)
  • Programmable pressure‐cooker (e.g., Biocare Medical Decloaking Chamber) or alternative heating device (e.g., water bath, microwave oven, vegetable steamer)
  • Rocker that can accommodate a staining dish
  • Low‐lint wipes (e.g., VWR, cat. no. 115‐0202)
  • Hydrophobic pen (Dako, cat. no. S2002)
  • Humidified chamber: commercial (e.g., StainTray, Simport, cat. no. M920) or prepared as in Figure
  • 37° and 60°C incubators
  • Aluminum foil
  • Glass coverslips (VWR, cat. no. 631‐9430)
  • Fluorescent confocal microscope with standard FITC filter (peak excitation, 490 nm; peak emission, 526 nm)
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Literature Cited

Literature Cited
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