In Vivo Immuno‐Spin Trapping: Imaging the Footprints of Oxidative Stress

Nicholas K.H. Khoo1, Nadiezhda Cantu‐Medellin2, Claudette St. Croix3, Eric E. Kelley4

1 Departments of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, 2 Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania, 3 Center for Biologic Imaging, University of Pittsburgh, Pittsburgh, Pennsylvania, 4 Department of Anesthesiology, University of Pittsburgh, Pittsburgh, Pennsylvania
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.42
DOI:  10.1002/0471142956.cy1242s74
Online Posting Date:  October, 2015
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


A plethora of disease processes are associated with elevated reactive species formation and allied reactions with biomolecules that alter cell signaling, induce overt damage, and promote dysfunction of tissues. Unfortunately, effective detection of reactive species in tissues is wrought with issues that significantly limit capacity for validating species identity, establishing accurate concentrations, and identifying anatomic sites of production. These shortcomings reveal the pressing need for new approaches to more precisely assess reactive species generation in vivo. Herein, we describe an in vivo immuno‐spin trapping method for indirectly assessing oxidant levels by detecting free radicals resulting from reaction of oxidants with biomolecules to form stable, immunologically detectable nitrone‐biomolecular adducts. This process couples the reactivity and sensitivity of an electron paramagnetic resonance spin trap with the resolution of confocal imaging to visualize the extent of cell and tissue oxidation and anatomic sites of production by detecting resultant free radical formation. © 2015 by John Wiley & Sons, Inc.

Keywords: 5,5‐dimethyl‐1‐pyrroline N‐oxide (DMPO); electron paramagnetic resonance (EPR); immuno‐spin trapping; reactive species; free radicals; oxidative stress

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: In Vivo Immuno‐Spin Trapping (IST)
  • Alternate Protocol 1: Anti‐DMPO Western Blot
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


Basic Protocol 1: In Vivo Immuno‐Spin Trapping (IST)

  • Laboratory mice with diet‐induced obesity (Khoo et al., )
  • 5,5‐dimethyl‐1‐pyrroline N‐oxide (DMPO) solution (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Liquid nitrogen
  • 2% (v/v) paraformaldehyde (PFA)/PBS: dilute 4% (v/v) PFA (Santa Cruz Biotechnology, cat. no. SC‐281692) with an equal volume of PBS
  • 30% (w/v) sucrose (Sigma, cat. no. S7903)
  • 2‐methylbutane (Fisher Cat No. O3551‐4)
  • PBB: 20% (v/v) serum (from the species in which secondary antibodies are made)/0.5% BSA (v/v)/PBS
  • Anti‐DMPO polyclonal antibody (ALX‐210‐530‐R050, Enzo Life Sciences)
  • Secondary antibody: Alexa Fluor 488 anti‐rabbit IgG (Life Technologies)
  • Hoechst stain: 1 mg Hoechst stain (Sigma, cat. no. B‐2883)/100 ml water
  • Gelvatol (see recipe)
  • Test tubes large enough for fixing and embedding samples, e.g., 15‐ml conical, polystyrene or glass test tubes (Fisher)
  • 250‐ml plastic beaker
  • Ice bucket, Styrofoam box, or equivalent
  • Filter paper
  • Forceps, small with blunt tips
  • Cryovials, cryopreservation bags, or pasteboard sliding boxes (EMS; of appropriate size
  • Glass microscope slides (e.g., Fisher brand Superfrost Plus, Fisher Cat. No.12‐550‐15) and cover glasses
  • Slide box
  • Additional reagents and equipment for injecting and euthanizing mice (Donovan and Brown, ) and performing cryosectioning (Watkins, ; unit 12.15), immunohistochemistry (Watkins, ; unit 12.16), and scanning confocal laser microscopy (Smith, )

Alternate Protocol 1: Anti‐DMPO Western Blot

  • Frozen organs or tissues from DMPO‐treated mice ( protocol 1Basic Protocol, step 3a)
  • Chelex‐treated PBS, pH 7.4: prepare using PBS ( appendix 2A) and Chelex 100 (Bio‐Rad) according to the manufacturer's directions
  • 100 μM diethylene triamine pentaacetic acid (DTPA; Sigma‐Aldrich)
  • BCA assay kit (Pierce)
  • 5% (v/v) BSA (Sigma)/TBST (see recipe)
  • TBST (see recipe)
  • 1:1000 rabbit anti‐DMPO polyclonal antibody (Enzo Life Sciences, cat. no. ALX‐210–530‐R050)/5% (v/v) BSA
  • 1:5000 goat anti‐rabbit IgG (Abnova, cat. no. PAB13665)/5% (v/v) BSA
  • ECL‐plus chemiluminescence kit (GE/Amersham)
  • Phosphine‐based reducing buffer (e.g., Bio‐Rad XT, cat. no. 161‐0792)
  • Homogenizers
  • Refrigerated centrifuge
  • Software for measuring gel staining density (e.g., UN‐SCAN‐IT; Silk Scientific)
  • Additional reagents and equipment for performing SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) and western blotting (Gallagher et al., )
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
  Chatterjee, S., Ehrenshaft, M., Bhattacharjee, S., Deterding, L.J., Bonini, M.G., Corbett, J., Kadiiska, M.B., Tomer, K.B., and Mason, R.P. 2009. Immuno‐spin trapping of a post‐translational carboxypeptidase B1 radical formed by a dual role of xanthine oxidase and endothelial nitric oxide synthase in acute septic mice. Free Radic. Biol. Med. 46:454‐461. doi: 10.1016/j.freeradbiomed
  Donovan, J. and Brown, P. 2006. Use of mouse, rat, hamster, and rabbit. Curr. Protoc. Immunol. 73:1.6.1‐1.6.10.
  Gallagher, S. Winston, S.E., Fuller, S.A., and Hurrell, J.G.R. 2008. Immunoblotting and immunodetection. Curr. Protoc. Mol. Biol. 83:10.8.1‐10.8.28.
  Gomez‐Mejiba, S.E., Zhai, Z., Della‐Vedova, M.C., Muñoz, M.D., Chatterjee, S., Towner, R.A., Hensley, K., Floyd, R.A., Mason, R.P., and Ramirez, D.C. 2014. Immuno‐spin trapping from biochemistry to medicine: Advances, challenges, and pitfalls. Focus on protein‐centered radicals. Biochim. Biophys. Acta 1840:722‐729. doi: 10.1016/j.bbagen.2013.04.039
  Kalyanaraman, B., Darley‐Usmar, V., Davies, K.J., Dennery, P.A., Forman, H.J., Grisham, M.B., Mann, G.E., Moore, K., Roberts, L.J., 2nd, and Ischiropoulos, H. 2012. Measuring reactive oxygen and nitrogen species with fluorescent probes: Challenges and limitations. Free Radic. Biol. Med. 52:1‐6. doi: 10.1016/j.freeradbiomed
  Khoo, N.K., Cantu‐Medline, N., Fleming, A.M., Champion, H.C., Devlin, J.E., Watkins, S., Mason, R.P., Freeman, B.A., and Kelley, E.E. 2012. Obesity‐induced tissue free radical formation: An immunospin trapping study. Free Radic. Biol. Med. 52:2312‐2319. doi: 10.1016/j.freeradbiomed
  Kojima, C., Ramirez, D.C., Tokar, E.J., Himeno, S., Drobna, Z., Styblo, M., Mason, R.P., and Waalkes, M.P. 2009. Requirement of arsenic biomethylation for oxidative DNA damage. J. Natl. Cancer Inst. 101:1670‐1681. doi: 10.1093/jnci/djp414
  Mason, R.P. 2004. Using anti‐5,5‐dimethyl‐1‐pyrroline N‐oxide (anti‐DMPO) to detect protein radicals in time and space with immuno‐spin trapping. Free Radic. Biol. Med. 36:1214‐1223. doi: 10.1016/j.freeradbiomed.2004.02.077.
  Narwaley, M., Michail, K., Arvadia, P., and Siraki, A.G. 2011. Drug‐induced protein free radical formation is attenuated by unsaturated fatty acids by scavenging drug‐derived phenyl radical metabolites. Chem. Res. Toxicol. 24:1031‐1039. doi: 10.1021/tx200016h
  Siraki, A.G., Deterding, L.J., Bonini, M.G., Jiang, J., Ehrenshaft, M., Tomer, K.B., and Mason, R.P. 2008. Procainamide, but not N‐acetylprocainamide, induces protein free radical formation on myeloperoxidase: A potential mechanism of agranulocytosis. Chem. Res. Toxicol. 21:1143‐1153. doi: 10.1021/tx700415b
  Smith, C.L. 2011. Basic confocal microscopy. Curr. Protoc. Neurosci. 56:2.2.1‐2.2.18.
  Stadler, K., Bonini, M.G., Dallas, S., Duma, D., Mason, R.P., and Kadiiska, M.B. 2008. Direct evidence of iNOS‐mediated in vivo free radical production and protein oxidation in acetone‐induced ketosis. Am. J. Physiol. Endocrinol. Metab. 295:E456‐E462. doi: 10.1152/ajpendo.00015
  Tarpey, M.M. and Fridovich, I. 2001. Methods of detection of vascular reactive species: Nitric oxide, superoxide, hydrogen peroxide, and peroxynitrite. Circ. Res. 89:224‐236.
  Tarpey, M.M., Wink, D.A., and Grisham, M.B. 2004. Methods for detection of reactive metabolites of oxygen and nitrogen: In vitro and in vivo considerations. Am. J. Physiol. Regul. Integr. Comp. Physiol. 286:R431‐R444. doi: 10.1152/ajpregu.00361.2003.
  Watkins, S. 2009a. Cryosectioning. Curr. Protoc. Cytom. 48:12.15.1‐12.15.7.
  Watkins, S. 2009b. Immunohistochemistry. Curr. Protoc. Cytom. 48:12.16.1‐12.16.10.
PDF or HTML at Wiley Online Library