Staining of Frozen and Formalin‐Fixed, Paraffin‐Embedded Tissues with Metal‐Labeled Antibodies for Imaging Mass Cytometry Analysis

Qing Chang1, Olga Ornatsky1, David Hedley2

1 Fluidigm Canada Inc., Markham, Ontario, 2 Division of Medical Oncology and Hematology, Princess Margaret Cancer Centre, Toronto, Ontario
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.47
DOI:  10.1002/cpcy.29
Online Posting Date:  October, 2017
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Abstract

This unit describes protocols for labeling tissue sections using combinations of metal‐tagged antibodies and an iridium‐containing DNA intercalator for analysis by imaging mass cytometry. Imaging mass cytometry (IMC) allows the labeling of up to 40 individual markers simultaneously using antibody cocktails. We discuss labeling of both cryostat sections and sections from formalin‐fixed, paraffin‐embedded (FFPE) tissue blocks. The protocols are similar to those used for optical microscopy techniques, while allowing much higher complexity of analysis. © 2017 by John Wiley & Sons, Inc.

Keywords: antibodies; FFPE; imaging mass cytometry; mass cytometry; proliferation

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Staining Frozen Sections with Metal‐Tagged Antibodies
  • Basic Protocol 2: Staining FFPE Sections with Metal‐Tagged Antibodies
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Staining Frozen Sections with Metal‐Tagged Antibodies

  Materials
  • Frozen tissue sections [tissue embedded in optimal cutting temperature (OCT) compound, snap frozen, sectioned on a microtome‐cryostat, section thickness user‐determined (normally 5 to 10 µm); see unit ; Watkins, ]
  • Acetone (Sigma‐Aldrich®, cat. no. 67‐64‐1)
  • Dulbecco's phosphate‐buffered saline (DPBS) without Ca or Mg (Thermo Fisher Scientific™, cat. no. 14190‐144)
  • Bovine serum albumin (BSA; Rockland, cat. no. BSA‐50)
  • Metal‐conjugated antibodies (Fluidigm; Table 12.47.1)
  • Triton™ X‐100 (Thermo Fisher Scientific, cat. no. 85111)
  • Intercalator‐Ir (Fluidigm®, cat. no. PN 201192B)
  • Toluidine blue O, 0.1% aqueous solution (Electron Microscopy Sciences, cat. no. 26074‐05)
Table 2.7.1   MaterialsMetal‐Conjugated Antibodies and Element‐Containing Reagents Used to Stain Cryostat Sections for Imaging Mass Cytometry Illustrated in Figure

Antibody/reagent Antibody clone Metal Fluidigm cat. no. Final concentration (µg/ml)
αSMA 1A4 141Pr 3141017D 1.25
Vimentin RV202 156Gd 3156023A 2.5
E‐cadherin 24E10 158Gd 3158021A 2.5
Pan‐keratin C11 162Dy 3162027A 1.25
β‐catenin D13A1 165Ho 3165027A 2.5
Ki‐67 B56 168Er 3168007B 2.5
Collagen I Goat polyclonal 169Tm 3169023D 1.25
Intercalator 191Ir, 193Ir 201192B 0.25 µM

  • PAP pen (Sigma‐Aldrich, cat. no. Z377821)
  • Hydration chamber: e.g., plastic slide box with lid, containing moist Kimwipes
  • Slide holder
  • Coplin jars
  • Microcentrifuge

Basic Protocol 2: Staining FFPE Sections with Metal‐Tagged Antibodies

  Materials
  • FFPE tissue sections [FFPE tissue blocks sectioned on a microtome, section thickness user‐determined, (normally 4 to 5 µm); see unit ; Watkins, ]
  • M‐xylene (Sigma‐Aldrich, cat. no. 185566‐1 liter)
  • Anhydrous ethyl alcohol (USP+, cat. no. 432526)
  • Antigen Retrieval Reagent–Basic, 10× (R&D Systems, cat. no. CTS013)
  • Dulbecco's phosphate‐buffered saline (DPBS) without Ca or Mg (Thermo Fisher Scientific, cat. no. 14190‐144)
  • Bovine serum albumin (BSA; Rockland, cat. no. BSA‐50)
  • Metal‐conjugated antibodies (Fluidigm; Table 12.47.1)
  • Triton X‐100 (Thermo Fisher Scientific, cat. no. 85111)
  • Intercalator‐Ir (Fluidigm, PN 201192B)
  • Toluidine blue O, 0.1% aqueous solution (Electron Microscopy Sciences, cat. no. 26074‐05)
  • Heating block
  • 50‐ml centrifuge tubes
  • PAP pen (Sigma‐Aldrich, cat. no. Z377821)
  • Hydration chamber: e.g., plastic slide box with lid, containing moist Kimwipes
  • Slide holder
  • Coplin jars
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Figures

Videos

Literature Cited

Literature Cited
   Bodenmiller, B. (2016). Multiplexed epitope‐based tissue imaging for discovery and healthcare applications. Cell Systems, 4, 225–238. doi: 10.1016/j.cels.2016.03.008.
   Chang, Q. , Ornatsky, O. I. , Siddiqui, I. , Laboda, A. , Baranov, V. I. , & Hedley, D. W. (2017). Imaging mass cytometry. Cytometry part A, 91, 160–169. doi: 10.1002/cyto.a.23053.
   Chang, Q. , Ornatsky, O. I. , Siddiqui, I. , Straus, R. , Baranov, V. I. , & Hedley, D. W. (2016). Biodistribution of cisplatin revealed by imaging mass cytometry identifies extensive collagen binding in tumor and normal tissues. Scientific Reports, 6, 36641. doi: 10.1038/srep36641.
   Giesen, C. , Wang, H. A. , Schapiro, D. , Zivanovic, N. , Jacobs, A. , Hattendorf, B. , … Bodenmiller, B. (2014). Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry. Nature Methods, 4, 417–422. doi: 10.1038/nmeth.2869.
   Ornatsky, O. I. , Lou, X. , Nitz, M. , Schafer, S. , Sheldrick, W. S. , Baranov, V. I. , … Tanner, S. D. (2008). Study of cell antigens and intracellular DNA by identification of element‐containing labels and metallointercalators using inductively coupled plasma mass spectrometry. Analytical Chemistry, 7, 2539–2547. doi: 10.1021/ac702128m.
   Watkins, S. (2009). Immunohistochemistry. Current Protocols in Cytometry, 48, 12.16.1–12.16.10. doi: 10.1002/0471142956.cy1216s48.
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