Isolation and Biochemical Characterization of Amyloid Plaques and Paired Helical Filaments

Agueda Rostagno1, Jorge Ghiso2

1 Department of Pathology, New York University, New York, New York, 2 Department of Psychiatry, New York University, New York, New York
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.33
DOI:  10.1002/0471143030.cb0333s44
Online Posting Date:  September, 2009
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Abstract

Extracellular deposits of amyloid fibrils in the form of parenchymal plaques and cerebrovascular lesions, as well as intracellular accumulation of paired‐helical filaments in the form of neurofibrillary tangles (NFT) in selected neuronal populations are the main neuropathologic hallmarks of Alzheimer's disease. Amyloid fibrils composed of polymeric structures of the amyloid‐β (Aβ) concentrate at the center of senile plaques and accumulate in the walls of cerebral blood vessels, exhibiting extensive Congo red/thioflavin S staining. Intraneuronal NFT are composed of building blocks of aberrantly hyperphosphorylated species of the microtubule‐associated protein tau, which accumulate in the perinuclear cytoplasm of vulnerable neurons in the form of paired helical filaments (PHF). This unit presents a variety of protocols for the isolation, biochemical analysis, and characterization of amyloid fibrils and neurofibrillary tangles. Curr. Protoc. Cell Biol. 44:3.33.1‐3.33.33. © 2009 by John Wiley & Sons, Inc.

Keywords: Alzheimer's disease; amyloid; neurofibrillar tangles; paired‐helical filaments; laser capture microdissection; immunoblotting; immunoprecipitation; mass spectrometry; amino acid sequence

     
 
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Table of Contents

  • Introduction
  • Amyloid/Pre‐Amyloid Purification
  • Basic Protocol 1: Extraction and Solubilization of Amyloid and Pre‐Amyloid Deposits for Subsequent Biochemical and Mass‐Spectrometry Studies
  • Alternate Protocol 1: Extraction of Amyloid Fibrils and Plaque Cores
  • Alternate Protocol 2: Extraction of Plaque Cores
  • Alternate Protocol 3: Extraction of Amyloid from Leptomeningeal Vessels and Microvessels
  • Alternate Protocol 4: Plaque Isolation by Laser Capture Microdissection
  • Microscopic Detection of Amyloid
  • Support Protocol 1: Aβ Immunohistochemistry
  • Support Protocol 2: Congo Red Staining
  • Support Protocol 3: Thioflavin S Staining
  • Support Protocol 4: Electron Microscopy
  • Biochemical Analysis of Amyloid in the Brain Extracts
  • Basic Protocol 2: Immunoprecipitation of Amyloid and Pre‐Amyloid Extracted Fractions
  • Support Protocol 5: Immunoblot Analysis
  • Support Protocol 6: Amino Acid Sequence Analysis
  • Purification and Characterization of Paired Helical Filaments
  • Basic Protocol 3: Isolation of PHF by Sedimentation Centrifugation
  • Alternate Protocol 5: An Alternative for Isolation of PHF by Sedimentation Centrifugation
  • Alternate Protocol 6: NFT Isolation by Laser Capture Microdissection
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Extraction and Solubilization of Amyloid and Pre‐Amyloid Deposits for Subsequent Biochemical and Mass‐Spectrometry Studies

  Materials
  • Frozen brain tissue
  • Tissue homogenization buffer 1 (THB‐1; see recipe)
  • 2% (w/v) SDS in 20 mM Tris⋅Cl, pH 7.4: prepared by diluting 1:5 a commercially available 10% SDS solution (BioRad) in 20 mM Tris⋅Cl, pH 7.4
  • 70% (v/v) formic acid: prepared by diluting 99% formic acid (Sigma) with de‐ionized water
  • Sterile dissecting instruments including:
    • Scalpel
    • Tweezers
    • Small spatula
  • Razor blades
  • Dounce glass homogenizer
  • 300‐ and 70‐µm Spectra/mesh nylon filters (Spectrum Laboratories)
  • 10.4‐ml polycarbonate bottles (Beckman) for ultracentrifugation (step 4)
  • XL100K ultracentrifuge (Beckman Coulter) equipped with a Beckman 70.1 Ti rotor, or equivalent
  • Ice bath
  • Vortex
  • 5417 refrigerated microcentrifuge (Eppendorf), or equivalent
NOTE: All solutions should be ice‐cold and procedures should be carried out on ice or inside a 4°C cold room.

Alternate Protocol 1: Extraction of Amyloid Fibrils and Plaque Cores

  Materials
  • Frozen brain tissue
  • Tissue homogenization buffer 2 (THB‐2 see recipe)
  • 1.0, 1.2, 1.4, 2.0 M sucrose gradient solutions (see recipe)
  • Dounce glass homogenizer
  • 100°C water bath
  • 300‐µm, 70‐µm, and 35‐µm Spectra/mesh nylon filters (Spectrum Laboratories)
  • Ultracentrifuge XL100K (Beckman Coulter) equipped with 70.1 and SW40 Ti rotors, or equivalent
  • 10.4‐ml polycarbonate bottles (Beckman) for ultracentrifugation (step 5)
  • Ultra clear tubes (14 × 95–mm; Beckman) for ultracentrifugation (step 7)
  • Additional reagents and equipment for dissecting gray matter ( protocol 1) and microscopic detection of amyloids using Congo red staining ( protocol 7), Thioflavin S staining ( protocol 8), and electron microscopy ( protocol 9)

Alternate Protocol 2: Extraction of Plaque Cores

  • Wash buffer (see recipe)
  • 1.2, 1.4, 1.6, 1.8 M sucrose gradient solutions (see recipe)
  • 100‐µm and 35‐µm Spectra/mesh nylon filters (Spectrum Laboratories)
  • J‐6B refrigerated centrifuge (Beckman Coulter)
  • XL100K Ultracentrifuge (Beckman Coulter, Fullerton, CA) equipped with SW40 Ti rotors, or equivalent
  • Ultra‐clear tubes (14 × 95–mm; Beckman) for gradient centrifugation (step 6)
  • Additional reagents and equipment for preparing brain homogenates ( protocol 2)

Alternate Protocol 3: Extraction of Amyloid from Leptomeningeal Vessels and Microvessels

  Materials
  • Cortical brain tissue (e.g., frontal lobe)
  • Tissue homogenization buffer 3 (THB‐3; see recipe)
  • Protease inhibitor cocktail (Complete; Roche Applied Science)
  • 26% (w/v) dextran (MW 65,000‐85,000) in THB‐2
  • Percoll (Sigma)
  • Dounce glass homogenizer
  • J‐6B refrigerated centrifuge (Beckman Coulter), or equivalent
  • 350‐, 150‐, and 70‐µm nylon mesh filters
  • 50‐ml polypropylene tubes (Fisher Scientific)
  • XL100K ultracentrifuge (Beckman Coulter) equipped with a Beckman SW40 Ti rotor, or equivalent (for Percoll gradient)
  • 15 × 95–mm ultraclear tubes (Beckman)

Alternate Protocol 4: Plaque Isolation by Laser Capture Microdissection

  Materials
  • Post‐mortem blocks of frontal or temporal cortex
  • Cryostat mounting medium (Tissue‐Tek O.C.T.; Jed Pella)
  • 2‐methylbutane (Fisher Scientific)
  • 75% ethanol
  • Thioflavin S (Sigma)
  • Graded ethanol solutions: 50%, 95%, and 100% (v/v) ethanol
  • Xylene
  • HM500OM cryotome (MICROM International), or equivalent
  • Uncoated and uncharged glass slides (Fisher Scientific)
  • Fluorescence microscope equipped with a Pixcell II laser capture device (Arcturus), or similar
  • CapSure Macro LCM cap (Arcturus)
  • Additional reagents and equipment for laser capture microdissection (Liao et al., )

Support Protocol 1: Aβ Immunohistochemistry

  Materials
  • 8‐µm thick Paraffin‐embedded brain tissue sections on glass microscopy slides
  • Xylene
  • 100%, 95%, and 75% ethanol
  • De‐ionized water
  • 98% (w/v) formic acid
  • Ready‐to‐use peroxidase blocking reagent (DakoCytomation)
  • Vecstatin ABC kit (Vector Laboratories) including:
    • Blocking reagent
    • Secondary antibody
    • Vectastatin ABC
  • Phosphate‐buffered saline (PBS; see recipe)
  • 4G8 and 6E10 mouse monoclonal anti‐Aβ antibodies (Covance)
  • Ready‐to‐use antibody diluent (DakoCytomation)
  • Tween 20
  • DAB Chromogen (DakoCytomation)
  • 3% hydrogen peroxide
  • Hematoxylin (DakoCytomation)
  • Cytoseal‐60 low‐viscosity mounting medium (Richard‐Allan Scientific)
  • 60°C oven
  • Paper towels
  • Olympus BX60 microscope, or equivalent

Support Protocol 2: Congo Red Staining

  Materials
  • Congo red (Sigma)
  • 80% (v/v) ethanol
  • Tissue samples
  • 1 M NaOH
  • 4% (v/v) paraformaldehyde in PBS
  • Deionized water
  • Whatman no. 1 filter paper
  • Positively charged microscope slides (Fisher Scientific)
  • Super Pap Pen (Invitrogen)
  • Olympus BX60 microscope equipped with crossed polarizers, or equivalent

Support Protocol 3: Thioflavin S Staining

  Materials
  • Amyloid‐containing tissue sections
  • 4% (v/v) paraformaldehyde in PBS
  • Thioflavin S (Sigma)
  • 80% (v/v) ethanol
  • Mounting medium for fluorescence microscopy (Vector Laboratories)
  • Olympus BX60 epi‐fluorescence microscope or equivalent

Support Protocol 4: Electron Microscopy

  Materials
  • Sample to be analyzed, e.g., resuspended fraction
  • Milli‐Q water (purified water with resistivity values at 25°C <18.2 Mho/cm), sterile
  • 1% uranyl acetate, pH 6.0 (Electron microscopy grade, Spi‐Chem)
  • 300 mesh, electron microscope cooper or nickel grids (Canemco & Marivac, Quebec)
  • Filter paper
  • CM12 Philips Transmission Electron Microscope or equivalent

Basic Protocol 2: Immunoprecipitation of Amyloid and Pre‐Amyloid Extracted Fractions

  Materials
  • Paramagnetic beads coated with goat anti‐mouse IgG (Dynabeads M‐280, Dynal/Invitrogen)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 4G8 and 6E10 monoclonal anti Aβ antibodies (Covance)
  • Blocking buffer: PBS containing 0.1% (w/v) bovine serum albumin (BSA; Sigma)
  • SDS‐OUT (Pierce Biotechnology)
  • 0.5 M Tris⋅Cl, pH 11.0
  • 10% 2‐mercaptoethanol (Sigma)
  • Milli‐Q water (Purified water with resistivity values at 25°C <18.2 Mho/cm)
  • 4:4:1 mixture of isopropyl alcohol/water/formic acid
  • 1.5‐ml microcentrifuge tubes
  • Eppendorf tube‐rack equipped with a removable slide‐out magnet (Dynal MPC‐S)
  • Mini labRoller bi‐directional rotator (Labnet International)
  • Savant SpeedVac concentrator (Global Medical Instrumentation)
  • Additional reagents and equipment for immunoblot assay ( protocol 11)

Support Protocol 5: Immunoblot Analysis

  Materials
  • Acrylamide stock: 40% acrylamide/Bis solution, 29:1 (3.3% C; BioRad)
  • Gel buffer: 3 M Tris⋅Cl, 0.3% (w/v) SDS, pH 8.45
  • Glycerol
  • Deionized water
  • 10% (w/v) ammonium persulfate (APS)
  • TEMED (N,N,N,N′‐tetra‐methyl‐ethylendiamine; BioRad)
  • Isopropanol
  • Samples to be analyzed
  • Tris‐tricine sample buffer (BioRad)
  • Cathode buffer: Tris‐Tricine‐SDS buffer 10× concentrate (Sigma)
  • Anode buffer (10× concentrate): 2 M Tris⋅Cl, pH 8.9
  • Transfer buffer: 10 mM 3‐cyclohexylamino‐1‐propanesulfonic acid (CAPS, Sigma), pH 11.0, containing 10% (v/v) methanol
  • Blocking solution: 5% non‐fat dry milk in PBS containing 0.1% Tween 20 (Sigma)
  • Monoclonal anti‐Aβ antibodies, 4G8 and 6E10 (Covance)
  • Tris‐buffered saline (Fisher Scientific) containing 0.1% Tween 20 (TBS‐T)
  • HRP‐labeled F(ab′)2 anti–mouse IgG (GE Lifesciences)
  • Western Blotting ECL Detection Substrate (SuperSignal West Pico, Pierce Biotechnology)
  • Hoeffer MiniVE Mini Vertical Electrophoresis Unit (Fisher Scientific)
  • Nitrocellulose membranes for chemiluminescence (Hybond ECL; GE Lifesciences), prewet
  • Hoeffer TE22 Mini Tank Blotting Unit (Fisher Scientific)
  • Orbital Shaker (Bellco Biotechnology) or equivalent
  • Film for chemiluminescence (Hyperfilm ECL, GE Lifesciences)
  • M35A X‐OMAT Film Processor (Eastman Kodak Company) or equivalent
  • Power supply Power Station 300 plus (Labnet International, Inc.) or equivalent

Support Protocol 6: Amino Acid Sequence Analysis

  Materials
  • 100% methanol
  • Milli‐Q water (Purified water with resistivity values at 25°C <18.2 Mho/cm)
  • Transfer buffer: 10 mM 3‐cyclohexylamino‐1‐propanesulfonic acid (CAPS, Sigma), pH 11.0, containing 10% (v/v) methanol
  • Sample to be sequenced
  • Protein stain solution: 0.125% (w/v) Coomassie Blue R‐250 in 50% methanol
  • Destaining solution: 50% methanol
  • Polyvinylidene difluoride membranes (PVDF; Immobilon‐P; Millipore)
  • Scalpel
  • Microcentrifuge tubes
  • 494 Procise Protein Sequencer (Applied Biosystems) or equivalent
  • Additional reagents and equipment for immunblot assay ( protocol 11)

Basic Protocol 3: Isolation of PHF by Sedimentation Centrifugation

  Materials
  • Frozen brain tissue enriched in NFT, as determined by standard immunohistochemistry
  • Tissue homogenization 4 (THB‐4; see recipe)
  • PHF extraction buffer (see recipe)
  • Sarkosyl (Sodium Lauroyl Sarcosinate)
  • RAB buffer (see recipe)
  • Sucrose gradient solutions (see recipe)
  • Protease Inhibitors Cocktail (Roche)
  • 2 M guanidine isothiocyanate
  • Dounce glass homogenizer
  • Ultracentrifuge XL100K (Beckman Coulter, Fullerton, CA) equipped with 70.1 and SW40 Ti rotors, or equivalent
  • Sonicator
  • Additional reagents and equipment for preparing a discontinuous sucrose gradient ( protocol 2)

Alternate Protocol 5: An Alternative for Isolation of PHF by Sedimentation Centrifugation

  Materials
  • Tissue
  • 0.32, 1, 1.05, 1.1, 1.15, 1.2, 1.8, 2, 2.2 M sucrose (see recipe)
  • 0.32 M sucrose‐equilibrating buffer (see recipe)
  • Sodium dodecyl sulfate (SDS)
  • Dounce homogenizer
  • J‐6B refrigerated centrifuge (Beckman Coulter), or equivalent
  • 150‐, 70‐, 35‐µm nylon mesh
  • 150‐ and 30‐ml tubes
  • Glass bead column (bead sizes 0.25 to 0.30‐mm and 0.45 to 0.5‐mm)
  • Additional reagents and equipment for transmission electron microscopy ( protocol 9)

Alternate Protocol 6: NFT Isolation by Laser Capture Microdissection

  Materials
  • Tissue sections
  • Acetone, cold
  • 0.1% Triton X‐100
  • 70% formic acid
  • Sonicator
  • Vacuum centrifugation system
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Literature Cited

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