Measurement of Oxygen Radicals and Lipid Peroxidation in Neural Tissues

Edward D. Hall1, Jeffrey M. Bosken1

1 Spinal Cord and Brain Injury Research Center, University of Kentucky, Lexington, Kentucky
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 7.17
DOI:  10.1002/0471142301.ns0717s48
Online Posting Date:  July, 2009
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Abstract

One of the most completely validated processes involved in secondary tissue damage following acute brain or spinal cord injury and in many chronic neurodegenerative diseases has to do with the pathological formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). These are generated by multiple mechanisms and give rise to highly reactive oxygen radicals that can damage neuronal, glial, and microvascular elements. Particular interest has centered upon oxygen radical‐induced, iron‐catalyzed lipid peroxidation (LP) as the principal mechanism of neuronal injury associated with oxygen radicals. Thus, there has been a growing interest in monitoring increased oxygen radical levels as an index of oxidative stress, as well as measuring markers of LP‐associated oxidative injury in in vitro and in vivo model systems and neurological patient samples. Accordingly, the purpose of this unit is to provide a variety of methods for the measurement of hydroxyl radical formation and/or LP in nervous tissue or biofluids.Curr. Protoc. Neurosci. 48:7.17.1‐7.17.51. © 2009 by John Wiley & Sons, Inc.

Keywords: reactive oxygen species; reactive nitrogen species; oxygen‐free radicals; lipid peroxidation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Salicylate Trapping and HPLC Assay of Hydroxyl Radical
  • Basic Protocol 2: Spectrophotometric Assay of Lipid‐Conjugated Dienes
  • Basic Protocol 3: HPLC Assay of Vitamin E
  • Basic Protocol 4: HPLC Assay of Glutathione
  • Basic Protocol 5: HPLC‐Chemiluminescence Assay of Lipid Hydroperoxides
  • Support Protocol 1: Xylenol Orange Determination of Hydroperoxide Content in Standards
  • Basic Protocol 6: Thiobarbituric Acid Assay of Malondialdehyde
  • Basic Protocol 7: HPLC Assay of Malondialdehyde Using UV Detection
  • Alternate Protocol 1: HPLC Assay of TBA‐Malondialdehyde Adduct Using Fluorescence Detection
  • Basic Protocol 8: GC/MS Determination and Quantification of 15‐F2T Isoprostane
  • Alternate Protocol 2: Immunoassay of F2‐Isoprostanes
  • Basic Protocol 9: Instrumental Detection and Quantification of F4‐Neuroprostanes
  • Basic Protocol 10: Immunoassay for 4‐Hydroxynonenal
  • Alternate Protocol 3: Immunohistochemical Detection of 4‐Hydroxynonenal
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Salicylate Trapping and HPLC Assay of Hydroxyl Radical

  Materials
  • HPLC‐grade solvents (EM Science):
    • Acetonitrile
    • Tetrahydrofuran
    • Water
    • Ethanol
    • 30% (v/v) methanol
  • Monochloroacetic acid (Aldrich)
  • 3,4‐Dihydroxycinnamic acid (3,4‐DHCA; Aldrich)
  • Phenylalanine
  • Mannitol
  • Deferoxamine (Ciba)
  • 2,3‐ and 2,5‐Dihydroxybenzoic acid (2,3‐ and 2,5‐DHBA; Aldrich)
  • Salicylate (Aldrich)
  • Tissue samples of interest: ∼500‐mg pieces, dissected and immediately frozen in liquid nitrogen, and stored at –80°C
  • Magnetic stir bar and plate
  • Vacuum
  • 0.5‐ and 1.5‐ml microcentrifuge tubes
  • Balance
  • Sonicator fitted with a microprobe tip (e.g., VirSonic 60 Ultrasonicator; Virtis Company)
  • Refrigerated centrifuge (e.g., Tomy MTX 150 equipped with a TMA‐11 rotor)
  • 250‐µl autosampler vials (e.g., Sun Brokers)
  • High‐performance liquid chromatograph (HPLC) equipped with:
    • Autosampler (e.g., Perkin‐Elmer ISS‐100) with 150‐µl vials
    • Refrigerated constant‐temperature circulating water bath capable of cooling to 4°C
    • HPLC pump capable of delivering a flow rate of 1.0 ml/min at pressures up to 4000 psi (e.g., Waters 510 LC)
    • Biophase ODS analytical HPLC column (4.6 × 250–mm, 5‐µm particle diameter, BAS)
    • Electrochemical detector (e.g., Bioanalytical Systems)
    • Programmable UV absorbance detector (e.g., Applied Biosystems 785A)
    • Data acquisition software (e.g., Waters Maxima 820)

Basic Protocol 2: Spectrophotometric Assay of Lipid‐Conjugated Dienes

  Materials
  • Rat liver, fresh or frozen
  • Sucrose/PBS/EDTA solution (see recipe; containing 0.1 M EDTA)
  • Methanol
  • Chloroform
  • Oxygen‐free nitrogen
  • Cyclohexane, spectrophotometric‐grade
  • Tissue homogenizer (blade homogenizer)
  • 40‐ml graduated, heavy‐walled, stoppered centrifuge tubes
  • 40° to 50°C water bath
  • Spectrophotometer with 1‐cm path‐length cuvette
NOTE: Commercially available solvents in this protocol can be used without further purification.

Basic Protocol 3: HPLC Assay of Vitamin E

  Materials
  • HPLC‐grade water
  • HPLC‐grade methanol (EM Science)
  • Pyridine (Mallinckrodt)
  • Sodium perchlorate (Sigma)
  • Helium
  • α‐Tocopherol (vitamin E; Aldrich)
  • HPLC‐grade ethyl acetate (EM Science)
  • Experimental animal
  • Liquid nitrogen
  • Vacuum filtration device
  • 47‐mm, 0.5‐µm Teflon filter (Type FH; Millipore)
  • 250‐µl autosampler vials (e.g., Sun Brokers)
  • 1.5‐ml microcentrifuge tubes
  • 1.5‐ml amber autosampler vials (e.g., National Scientific)
  • Homogenizer
  • Sonicator fitted with a microprobe tip (e.g., VirSonic 60 Ultrasonicator; Virtis Company)
  • Refrigerated centrifuge (e.g., Tomy MTX 150 equipped with a TMA‐11 rotor)
  • 12 × 75–mm polypropylene culture tubes (e.g., Falcon)
  • High‐performance liquid chromatograph (HPLC) equipped with:
    • Autosampler (e.g., Perkin‐Elmer ISS‐100)
    • Refrigerated constant‐temperature circulating 4°C water bath
    • Inertsil C8 column (3.0 × 250–mm, 5‐µm particle size; Metachem Technologies)
    • Inertsil C8 guard column (10 × 4.3–mm, 5‐µm particle size; Metachem Technologies)
    • 3‐mm precolumn inlet filter (Rheodyne model 7335)
    • HPLC pump (e.g., Waters 510 LC pump or any pump capable of delivering a flow rate of 1.0 ml/min at pressures up to 4000 psi)
    • Electrochemical detector with a glassy carbon electrode and Ag/AgCl reference electrode, or any equivalent detector capable of oxidation at 700 mV (e.g., Waters 460)
    • Scanning fluorescence detector (Waters 470) or other detector capable of reading emission at 340 nm and excitation at 290 nm
    • Data acquisition system (Waters Millennium 2010 version 2.1 or equivalent)

Basic Protocol 4: HPLC Assay of Glutathione

  Materials
  • 0.15 M sodium acetate, pH 7.0 (see recipe)
  • Methanol, reagent grade (EM Science)
  • Helium
  • Reduced glutathione (GSH)
  • 10 mM HCl, ice cold
  • Oxidized glutathione (GSSG)
  • 5 mg/ml OPA solution (see recipe)
  • 100 mM sodium phosphate buffer, pH 7.0 ( appendix 2A)
  • Tissue of interest: 20‐ to 40‐mg samples stored at –70°C
  • 25 mM sodium phosphate buffer, pH 6.0 ( appendix 2A), ice cold
  • 100 mM Tris⋅Cl, pH 8.5 ( appendix 2A)
  • 25 and 50 mM dithiothreitol (DTT; Sigma)
  • 2 mM N‐ethylmaleimide (NEM; Sigma)
  • 2.5% (w/v) 5‐sulfosalicylic acid (SSA; Sigma)
  • Vacuum filtration unit
  • 0.5‐µm Teflon filter (Type FH; Millipore)
  • Sonicator fitted with a microprobe tip (e.g., VirSonic 60 Ultrasonicator; Virtis Company)
  • Refrigerated centrifuge (e.g., Tomy MTX 150 equipped with a TMA‐11 rotor)
  • High‐performance liquid chromatograph (HPLC) equipped with:
    • Autosampler equipped with a heater/cooler to maintain constant sample compartment temperature at 4°C, and carousel capable of holding up to 96 autosampler vials (e.g., Waters 717)
    • Analytical C18 reversed‐phase ODS column (25 cm × 4.6 mm; 5‐µm particle size)
    • RP18 guard column
    • HPLC pump (e.g., Waters 510 LC pump or any pump capable of delivering a flow rate of 1.0 ml/min at pressures up to 4000 psi)
    • Scanning fluorescence detector or other detector capable of reading emission at 420 nm and of excitation at 340 nm (e.g., Waters 470)
    • Data acquisition system (Waters Millennium 2010, version 2.1 or equivalent)
  • 1‐ml autosampler vials containing low‐volume inserts with springs and caps (Waters)

Basic Protocol 5: HPLC‐Chemiluminescence Assay of Lipid Hydroperoxides

  Materials
  • HPLC‐grade chloroform (EM Science)
  • HPLC‐grade methanol (EM Science)
  • 40 mM monobasic potassium phosphate
  • 25 mg/ml phosphocholine (PC; Avanti Polar Lipids)
  • 25 mg/ml phosphoethanolamine (PE; Avanti Polar Lipids)
  • Methylene blue
  • Appropriate standards (e.g., 15(S)‐HPEPE, 13(S)‐HPODE, or 9(S)‐HPODE, Biomol Research Laboratories)
  • 1% (v/v) butylated hydroxytoluene (BHT; Sigma)/methanol
  • Tissue samples, dissected and immediately frozen in liquid nitrogen, stored at –80°C
  • 0.15 M saline: 0.9% (w/v) NaCl in HPLC‐grade water
  • Nitrogen
  • 2:1 and 1:9 (v/v) chloroform/methanol, freshly prepared
  • Chemiluminescent (CL) cocktail (see recipe)
  • Vacuum
  • 200‐ml beaker
  • UV light source
  • High‐performance liquid chromatograph (HPLC) equipped with:
    • 3‐ml Supelclean LC‐Si column (Supelco)
    • SIL‐LC‐Si analytical HPLC column (25 × 4.6–mm, 5‐µm particle size; Supelco)
    • Guard column (e.g., Supelguard LC‐Si; Supelco)
    • HPLC pump capable of delivering 1 ml/min at pressures up to 4000 psi (e.g., Waters 600)
    • Autosampler at 4°C and containing a carousel capable of holding up to 96 autosampler vials (e.g., Waters 717)
    • Postcolumn mixing tee (e.g., ISCO 500D syringe pump)
    • Chemiluminescence (CL) detector (e.g., Jasco model no. 825‐CL)
    • Data acquisition system (Waters Millennium 2010, or equivalent)
  • 4‐ml amber glass vials with caps
  • 1.5‐ml microcentrifuge tubes
  • Sonicator fitted with a microprobe tip (e.g., VirSonic 60 Ultrasonicator; Virtis Company)
  • Refrigerated centrifuge (e.g., Tomy MTX 150 equipped with a TMA‐11 rotor)
  • Pasteur pipets
  • Reacti‐therm heating module/evaporating unit (Pierce)
  • Autosampler vials containing low‐volume inserts with springs and caps (Waters)
  • Additional reagents and equipment for xylenol orange assay (see protocol 6)

Support Protocol 1: Xylenol Orange Determination of Hydroperoxide Content in Standards

  Materials
  • Degassed water
  • Ferrous ammonium sulfate (FAS; see recipe)
  • Argon
  • Methanol
  • Butylated hydroxytoluene (BHT; Sigma)
  • Concentrated (18 M) H 2SO 4
  • Xylenol orange (Aldrich)
  • Standards to be tested (see protocol 5)
  • 1.5‐ml microcentrifuge tubes
  • Pasteur pipets
  • 125‐ml container
  • Spectrophotometer and cuvettes

Basic Protocol 6: Thiobarbituric Acid Assay of Malondialdehyde

  Materials
  • Biological sample: 1.0 to 2.0 mg/ml membrane protein or 0.1 to 0.2 mM lipid phosphate
  • TCA/TBA/HCl solution (see recipe)
  • Boiling water bath
  • Centrifuge
  • Spectrophotometer and cuvettes

Basic Protocol 7: HPLC Assay of Malondialdehyde Using UV Detection

  Materials
  • Tris base
  • HPLC‐grade water (EM Science)
  • 1 N HCl
  • HPLC‐grade acetonitrile (EM Science)
  • 0.1 mM H 2O 2
  • Malonaldehyde bis(diethylacetal) (also known as 1,1,3,3‐tetraethoxypropane or TEP; Aldrich)
  • 10 mM potassium phosphate buffer, pH 7.0 ( appendix 2A)
  • Frozen tissue samples: dissected and immediately frozen in liquid nitrogen, stored at –80°C
  • 40 mM Tris⋅Cl, pH 7.4 ( appendix 2A)
  • 10 mM FAS solution (see recipe)
  • 0.02% (v/v) butylated hydroxytoluene (BHT; Sigma) in acetonitrile
  • Vacuum filtration device
  • 47‐mm, 0.5‐µm Teflon filter (Type FH; Millipore)
  • 250‐ml glass‐stoppered flask
  • Parafilm
  • 37° and 50°C water baths
  • 10‐ml volumetric flask
  • UV spectrophotometer (e.g., Beckman model DU‐7500 diode array)
  • 1.5‐ml microcentrifuge tubes
  • Sonicator fitted with a microprobe tip (e.g., VirSonic 60 Ultrasonicator; Virtis Company)
  • Refrigerated centrifuge (e.g., Tomy MTX 150 fitted with a TMA‐11 rotor)
  • 1‐ml autosampler vials containing low‐volume inserts with springs and caps (Waters)
  • High‐performance liquid chromatograph (HPLC) equipped with:
    • Autosampler configured with a thermostatted sample compartment held at 4°C and a carousel capable of holding up to 96 autosampler vials (e.g., Waters 717)
    • Zorbax SB‐CN analytical HPLC column (4.6 × 25–cm, 5‐µm particle size; MAC‐MOD Analytical)
    • Zorbax SB‐CN guard column (4.0 × 12.5–mm, 5‐µm particle size) fitted with PEEK‐encapsulated frit‐gasket (MAC‐MOD Analytical)
    • HPLC pump capable of delivering 0.9 ml/min at pressures up to 4000 psi (e.g., Waters 600)
    • Programmable UV absorbance detector (e.g., Applied Biosystems 785A)
    • Data acquisition system (Waters Millennium 2010 version 2.1 or equivalent)

Alternate Protocol 1: HPLC Assay of TBA‐Malondialdehyde Adduct Using Fluorescence Detection

  • 0.9% (w/v) NaCl
  • TBA/acetate/DTPA solution (see recipe)
  • 5% (v/v) butylated hydroxytoluene (BHT) in 99% (v/v) ethanol
  • 0.2‐ and 0.45‐µm filters (Millipore)
  • Water bath capable of maintaining temperatures up to 100°C
  • 150 × 4.6–mm Inertsil ODS‐2 analytical HPLC column (MetaChem Tech)
  • 50 × 4.0–mm guard column (MetaChem Tech)
  • HPLC pump capable of delivering a rate of 1 ml/min
  • Fluorescence detector with excitation at 515 nm and emission at 553 nm

Basic Protocol 8: GC/MS Determination and Quantification of 15‐F2T Isoprostane

  Materials
  • 15 F 2t‐isoprostane‐d 4 (8‐iso prostaglandin F α‐d 4; Cayman Chemical cat. no. 316350) in methyl acetate
  • 1 mg/ml 15 F 2t‐isoprostane (8‐iso prostaglandin F α; Cayman Chemical cat. no. 16350) in absolute ethanol
  • Absolute ethanol USP
  • Sample fluid (e.g., serum, plasma, or cerebrospinal fluid)
  • 0.1 M HCl or KOH
  • Butylhydroxytoluene (BHT) or 2,6‐di‐tert‐butyl‐4‐methylphenol (Sigma‐Aldrich cat. no. B1378)
  • Methanol, HPLC grade
  • pH 3.0 solution: prepare a 1 mM HCl solution and adjust pH to 3.0 with 0.1 M HCl or KOH
  • Ethyl acetate, HPLC grade
  • Heptane, HPLC grade
  • Nitrogen
  • Pentafluorobenzylbromide (PFBB; Sigma‐Aldrich, cat. no. 101052) made up to 10% solution in acetonitrile
  • 10% diisopropylethylamine (N,N‐diisopropylethylamine; DIPE; Sigma‐Aldrich cat. no. D3887) in acetonitrile
  • Chloroform
  • TLC standard (prostaglandin F , methyl ester)
  • Phosphomolybdic acid spray solution (Sigma‐Adrich cat. no. P4869)
  • N,O‐Bis[trimethylsilyl]trifluoroacetamide (BSTFA; Pierce Biotechnology cat. no. 38830)
  • 13 × 100–mm tubes
  • C 18 solid phase extraction columns (Waters Corporation cat. no. WAT020805)
  • 30‐ and 50‐ml beakers
  • Silica solid phase extraction columns (Waters Corporation cat. no. WAT020810)
  • Speedisk Positive Pressure Processor 48 (J.T. Baker), optional
  • 10‐ml centrifuge tubes
  • 37°C heating block
  • Whatman Partisil 4 channel TLC plates (Whatman cat. no. 4865‐621 or Fisher Scientific cat. no. 05‐713‐327)
  • 150°C oven
  • Desiccator
  • Hair dryer
  • Chamber large enough to hold TLC plates vertically
  • Heating plate
  • Razor blades
  • 1.5‐ml microcentrifuge tubes
  • 10‐ml borosilicate glass centrifuge tubes
  • 50°C water bath
  • GC/MS‐NICI instrument containing a 30‐m, DB‐5 column (0.25 mm × 0.25 µm)

Alternate Protocol 2: Immunoassay of F2‐Isoprostanes

  Materials
  • Sample: cell cultures or tissues stored at –80°C
  • HPLC‐grade solvents (EM Science):
    • Absolute ethanol, ice cold
    • Hexane
    • Ethyl acetate
    • Methanol
  • 100 mM sodium phosphate buffer, pH 7.2, prepared with deionized water
  • 8‐Isoprostane EIA kit (8‐iso‐PGF ; Cayman Chemical) containing:
    • Precoated (mouse anti‐rabbit IgG) EIA 96‐well strip plates
    • 96‐well cover sheets
    • Tween 20
    • EIA tracer dye
    • EIA antiserum dye
    • Blue EIA kit box (96 well)
    • Ellman's reagent
    • EIA buffer concentrate (10×)
    • Wash buffer concentrate (400×)
    • 8‐Isoprostane AChE tracer
    • 8‐Isoprostane EIA antiserum
    • 8‐Isoprostane EIA standard
  • Polytron tissue homogenizer
  • Refrigerated microcentrifuge
  • C 18 Sep‐Pak columns (Waters)
  • 1.5‐ml microcentrifuge tubes
  • Vacuum centrifuge
  • Platform shaker
  • Microplate reader (e.g., Techan SLT Spectra) with microplate analysis software (e.g., DeltaSOFT II; Biometallics)

Basic Protocol 9: Instrumental Detection and Quantification of F4‐Neuroprostanes

  Materials
  • 15 F 2t‐isoprostane‐d 4 (8‐iso prostaglandin F α‐d 4; Cayman Chemical, cat. no. 316350)
  • 15 F 2t‐isoprostane (8‐iso prostaglandin F α; Cayman Chemical, cat. no. 16350)
  • Tissue sample (fresh or ≤1 year at −80°C; >30 mg)
  • Folch solution: 3:1 (v/v) chloroform/methanol containing 0.005% BHT, stored up to 6 months at −20°C, ice cold
  • Nitrogen
  • 0.9% NaCl
  • Methanol, HPLC grade
  • Butylated hydroxytoluene (BHT; Sigma)
  • Potassium hydroxide (KOH)
  • 11.8 M formic acid
  • pH 3.0 solution: prepare 1 mM HCl and adjust pH to 3.0 with 0.1 M HCl or deionized water
  • C 18 solid phase extraction columns (Waters, cat. no. WAT020805)
  • Heptane, HPLC grade
  • Ethyl acetate, HPLC grade
  • Sodium sulfate
  • Silica solid phase extraction columns (Waters, cat. no. WAT020810)
  • Pentafluorobenzylbromide (PFBB; Sigma‐Aldrich, cat. no. 101052)
  • Diisopropylethylamine (N,N‐diisopropylethylamine; DIPE; Sigma‐Aldrich, cat. no. D3887)
  • Whatman Partisil 4 channel TLC plates (Whatman, cat. no. 4865‐621 or Fisher Scientific, cat. no. 05‐713‐327)
  • Ethanol USP, absolute
  • Chloroform, HPLC grade
  • TLC standard (prostaglandin F , methyl ester)
  • Phosphomolybdic acid spray solution (Sigma‐Adrich, cat. no. P4869)
  • N,O‐Bis[trimethylsilyl]trifluoroacetamide (BSTFA; Pierce Biotechnology, cat. no. 38830)
  • 10‐ and 40‐ml centrifuge tubes
  • Handheld tissue homogenizer (e.g., PowerGen model 35, Fisher Scientific)
  • Centrifuge
  • 37°C incubator/heating block
  • Speedisk Positive Pressure Processor 48 (J.T. Baker), optional
  • 30‐ and 50‐ml beakers
  • Hair dryer
  • Heating plate
  • Razor blades
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge
  • 50°C water bath
  • Target vials
  • GC/MS‐NICI instrument containing a 30‐m DB‐5 column (0.25 mm × 0.25 µm)
  • Injector liner, e.g., Restek Open Top Drilled Uniliner (hole on top; Restek)

Basic Protocol 10: Immunoassay for 4‐Hydroxynonenal

  Materials
  • Protease inhibitor (PI) buffer: add 1 protease inhibitor tablet (Roche Applied Science cat. no. 1 836 153) to every 10 ml of Triton lysis buffer (see recipe), prepare immediately before use and keep chilled
  • Tissue
  • Bio‐Rad DC protein assay kit reagents (Bio‐Rad Laboratories Reagent A cat. no. 500‐0113; Reagent B cat. no. 500‐0114; Reagent S cat. no. 500‐0115)
  • TBS (see recipe)
  • Blocking buffer: dissolve 5 g of dry non‐fat milk in TBS (see recipe) to a final volume of 100 ml
  • Primary antibody for 4‐HNE, rabbit anti‐hydroxynonenal (HNE) (Alpha Diagnostics International cat. no. HNE11‐S)
  • Tris‐buffered saline with Tween 20, pH 8.0 (TBST; Sigma‐Aldrich cat. no. T‐9039)
  • Secondary antibody for 4‐HNE and 3‐NT IRDye800CW goat anti‐rabbit IgG (Rockland Immunochemicals, cat. no. 611‐131‐122)
  • 1.5‐ or 2.0‐ml centrifuge tubes
  • Sonicator
  • Refrigerated centrifuge
  • Slot blot apparatus
  • Nitrocellulose/filter paper sandwiches (Bio‐Rad Laboratories cat. no. 162‐0233)
  • Bio‐Dot SF filter paper (Bio‐Rad Laboratories cat. no. 162‐0161)
  • Blotting paper
  • 40°C oven
  • LI‐COR Odyssey imager or equivalent

Alternate Protocol 3: Immunohistochemical Detection of 4‐Hydroxynonenal

  Materials
  • 35‐µm (or other, as appropriate) sections of brain or spinal cord tissue
  • 10× (1 M) phosphate buffered saline (PBS; Fisher Scientific cat. no. BP399) dilute with deionized water to yield 0.2 M PBS
  • Sodium borohydride (NaBH 4)
  • 0.1 M 3‐(N‐morpholino)propanesulfonic acid hemisodium salt, pH 8.0 (MOPS, Sigma‐Adrich cat. no. M0289)
  • 0.3% H 2O 2 in PBS: add 1 part 30% hydrogen peroxide to 99 parts 0.2 M PBS; prepare fresh just before use
  • Blocking buffer (see recipe), prepared fresh
  • Primary antibody for HNE, rabbit anti‐4‐HNE (Calbiochem, EMD Chemicals cat. no. 393207)
  • Secondary antibody (biotinylated goat anti‐rabbit secondary antibody)
  • Secondary antibody kit, ABC‐AP kit (Vector Laboratories, cat. no. AK‐5001)
  • 0.1 M Tris⋅Cl, pH 8.2 ( appendix 2A)
  • Vector Blue Kit (Vector Laboratories, cat. no. SK‐5300) containing:
    • Reagent 1
    • Reagent 2
    • Reagent 3
  • Nuclear fast red (Vector Laboratories, cat. no. H‐3403)
  • Permount (Fisher Scientific, cat. no. SP15‐100)
  • 12‐well plates and tissue wells (netwells, Corning cat. no. 07‐200‐211)
  • Paint brush
  • Platform rocker
  • Aluminum foil
  • Microscope slides
  • Coverslips
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Figures

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Literature Cited

Literature Cited
   Althaus, J.S., Andrus, P.K., Hall, E.D., and VonVoigtlander, P.F. 1995. Improvements in the salicylate trapping method for measurement of hydroxyl radical levels in brain. In Central Nervous System Trauma: Research Techniques, Vol. 4 of Membrane‐Linked Diseases (S. Ohnishi and T. Ohnishi, eds.) pp. 437‐444. CRC Press, Boca Raton, Fla.
   Beckman, J.S. 1991. The double‐edged role of nitric oxide in brain function and superoxide‐mediated injury. J. Devel. Physiol. 15:53‐59.
   Buege, J.A. and Aust, S.D. 1978. Microsomal Lipid Peroxidation. Meth. Enzymol. 52:302‐310.
   Bull, A.W. and Marnett, L.J. 1985. Determination of malondialdehyde by ion‐pairing high‐performance liquid chromatography. Anal. Biochem. 149:284‐290.
   Chiueh, C.C., Krishna, G., Tulsi, P., Obata, T., Lang, K., Huang, S.J., and Murphy, D.L. 1992. Intracranial microdialysis of salicylic acid to detect hydroxyl radical generation through dopamine autoxidation in the caudate nucleus: Effect of MPP+. Free Radical Biol. Med. 13:581‐583.
   Cini, M., Fariello, R.G., Bianchetti, A., and Moretti, A. 1994. Studies on lipid peroxidation in rat brain. Neurochem. Res. 19:283‐288.
   Deng, Y., Thompson, B.M., Gao, X., and Hall, E.D. 2007. Temporal relationship of peroxynitrite‐induced oxidative damage, calpain‐mediated cytoskeletal degradation and neurodegeneration after traumatic brain injury. Exp. Neurol. 205:154‐165.
   Floyd, R.A., Watson, J., and Wong, P.K. 1984. Sensitive assay of hydroxyl free radical formation utilizing high pressure liquid chromatography with electrochemical detection of phenol and salicylate hydroxylation products. J. Biochem. Biophys. Methods 10:221‐235.
   Fukunaga, K., Suzuki, T., and Takama, K. 1993. Highly sensitive high‐performance liquid chromatography for the measurement of malondialdehyde in biological samples. J. Chromatogr. 621:77‐81.
   Fukunaga, K., Takama, K., and Suzuki, T. 1995. High performance liquid chromatographic determination of plasma malondialdehyde level without a solvent extraction procedure. Anal. Biochem. 230:20‐23.
   Gurney, M.E., Cutting, F.B., Zhai, P., Doble, A., Taylor, C.P., Andrus, P.K., and Hall, E.D. 1996. Antioxidants and inhibitors of glutamatergic transmission have therapeutic benefit in a transgenic model of familial amyotrophic lateral sclerosis. Ann. Neurol. 39:147‐157.
   Hall, E.D. 2009. Handbook of Neurochemistry and Molecular Neurobiology (A. Lajtha, N. Banik, and S.K. Ray, eds.) pp. 209‐212. Springer Science and Business Media, New York.
   Hall, E.D. and Braughler, J.M. 1993. Free radicals in CNS injury. In Molecular and Cellular Approaches to the Treatment of Neurological Disease (S.G. Waxman, ed.) pp. 81‐105. Raven Press, New York.
   Hall, E.D., Yonkers, P.A., Horan, K.L., and Braughler, J.M. 1989. Correlation between attenuation of post‐traumatic spinal cord ischemia and preservation of spinal tissue vitamin E by the 21‐aminosteroid lipid peroxidation inhibitor U‐74006F: Evidence for an in vivo antioxidant mechanism. J. Neurotrauma 6:169‐176.
   Hall, E.D., Pazara, K.E., and Braughler, J.M. 1991. Effects of tirilazad mesylate on post‐ischemic brain lipid peroxidation and recovery of extracellular calcium in gerbils. Stroke 22:361‐366.
   Hall, E.D., Andrus, P.K., Althaus, J.S., and VonVoigtlander, P.F. 1993. Hydroxyl radical production and lipid peroxidation parallel selective post‐ischemic vulnerability in gerbil brain. J. Neurosci. Res. 34:107‐112.
   Hall, E.D., Detloff, M.R., Johnson, K., and Kupina, N.C. 2004. Peroxynitrite‐mediated protein nitration and lipid peroxidation in a mouse model of traumatic brain injury. J. Neurotrauma 21:9‐20.
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