Cyclic Immunofluorescence (CycIF), A Highly Multiplexed Method for Single‐cell Imaging

Jia‐Ren Lin1, Mohammad Fallahi‐Sichani1, Jia‐Yun Chen1, Peter K. Sorger1

1 HMS LINCS Center, Laboratory of Systems Pharmacology, Department of Systems Biology, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/cpch.14
Online Posting Date:  December, 2016
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Abstract

Cyclic Immunofluorescence (CycIF) is a public‐domain method for performing highly multiplexed immunofluorescence imaging using a conventional epifluorescence microscope. It uses simple reagents and existing antibodies to construct images with up to 30 channels by sequential 4‐ to 6‐channel imaging followed by fluorophore inactivation. Three variant methods are described, the most generally useful of which involves staining fixed cells with antibodies directly conjugated to Alexa Fluor dyes and imaging in four colors, inactivating fluorophores using a mild base in the presence of hydrogen peroxide and light, and then performing another round of staining and imaging. Cell morphology is preserved through multiple rounds of CycIF, and signal‐to‐noise ratios appear to increase. Unlike antibody‐stripping methods, CycIF is gentle and optimized for monolayers of cultured cells. A second protocol involves indirect immunofluorescence and a third enables chemical inactivation of genetically encoded fluorescent proteins, allowing multiplex immunofluorescence to be combined with live‐cell analysis of cells expressing fluorescent reporter proteins. © 2016 by John Wiley & Sons, Inc.

Keywords: CycIF; immunofluorescence; high‐content imaging; multiplexing; systems biology

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Type I CycIF: Using Antibodies Directly Conjugated to Alexa Fluorophores
  • Alternate Protocol 1: Type II CycIF: Cyclic Immunofluorescence With Both Non‐Conjugated Antibodies and Conjugated Antibodies
  • Alternate Protocol 2: Type III CycIF: Cyclic Immunofluorescence for Cells Expressing Fluorescent Proteins (FPS)
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Type I CycIF: Using Antibodies Directly Conjugated to Alexa Fluorophores

  Materials
  • Cells of interest in Corning 96‐well plates (clear‐bottom, black; Sigma‐Aldrich, cat. no. CLS3603)
  • 4% PFA: dilute 16% paraformaldehyde (PFA; Electron Microscopy Sciences, cat. no. 15710) 1:4 in 1× PBS
  • 1× PBS: dilute 20× phosphate‐buffered saline (PBS; Santa Cruz Biotech, cat. no. SC‐362299) 1:20 in MilliQ water
  • Methanol (99.9%; Fisher Scientific, cat. no. AC61009), store in −20°C
  • Odyssey blocking buffer (LICOR Odyssey, cat. no. 927‐40125)
  • Antibodies against antigens of interest, directly conjugated to Alexa Fluor dyes; many such antibodies are available and those tested with CycIF to date are listed in Table 14.0.110 mg/ml Hoechst 33342 (Invitrogen, cat. no. H3570)
  • Fluorophore inactivation solution for Alexa dyes: 3% (v/v) H 2O 2 (1/10 dilution from 30% stock; Sigma‐Aldrich, cat. no. 216763)/20 mM NaOH (Sigma‐Aldrich, cat. no. S5881) in 1× PBS
  • Biotek EL406 washer/dispenser (alternatively, multichannel pipettor)
  • Microscope for imaging multi‐well plates (e.g., GE Cytell Cell Imaging System)
  • Lamp to promote fluorophore inactivation: a simple table lamp equipped with a Lithona 211E71 LED light (UCLD 24 WH, color temperature: 3000k with CRI:83) has proven acceptable (the peak wavelength of the light source is at 630 nm with second peak at 540 nm and third peak at 450 nm; Fig.  )

Alternate Protocol 1: Type II CycIF: Cyclic Immunofluorescence With Both Non‐Conjugated Antibodies and Conjugated Antibodies

  Additional Materials (also see Basic Protocol 1)
  • Primary antibodies against antigens of interest, unconjugated
  • Secondary antibodies conjugated to Alexa Fluor dyes (e.g., ThermoFisher/Invitrogen goat anti‐rabbit IgG Alexa Fluor 488; cat. no. A11008)
  • Fluorophore inactivation solution for indirect IF using Alexa dyes: 4.5% H 2O 2/25 mM NaOH in 1× PBS

Alternate Protocol 2: Type III CycIF: Cyclic Immunofluorescence for Cells Expressing Fluorescent Proteins (FPS)

  Additional Materials (also see protocol 1Basic Protocol)
  • 1 M HCl (dilute from 12 M solution; Sigma‐Aldrich, cat. no. 258148)
  • Fluorophore inactivation solution for GFP/YFP/mCherry fusion proteins: 3% H 2O 2 (1/10 dilution from 30% stock), 20 mM HCl (1/100 dilution from 2 M stock) in 1× PBS
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Figures

Videos

Literature Cited

Literature Cited
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