Immunohistochemical Localization of Proteins in the Nervous System

Laura A. Volpicelli‐Daley1, Allan Levey1

1 Emory University, Atlanta, Georgia
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 1.2
DOI:  10.1002/0471142301.ns0102s25
Online Posting Date:  February, 2004
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Abstract

The immunohistological methods described in this unit can be used to determine the precise localization of neurochemicals, receptors, and proteins throughout the nervous system. Determining the localization of a protein within defined brain nuclei and neuronal cell populations can provide important clues regarding its potential function. Immunoperoxidase reactions and light microscopy are commonly used to visualize the distribution of a single primary antibody directed to an antigen of interest. Double‐labeling immunofluorescence and confocal microscopy techniques detect the localization of one protein relative to another protein and allow analysis of colocalization at a cellular and subcellular level. The colocalization of two proteins can also be quantified, allowing analysis of the extent of overlap between two labeled markers and measurements of changes in the localization of one protein relative to another following drug treatment or in animals that have been genetically modified. The theoretical limit of resolution of confocal microscopy is 0.1 to 0.2 µm.

Keywords: Immunohistochemistry; double‐labeling; immunofluorescence; quantitation of confocal images; electron microscopy

     
 
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Table of Contents

  • Basic Protocol 1: Immunohistochemistry for Light Microscopy
  • Basic Protocol 2: Double‐Labeling Immunofluorescence and Quantitation of Confocal Images
  • Basic Protocol 3: Pre‐Embedding Immunohistochemistry for Electron Microscopy
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Immunohistochemistry for Light Microscopy

  Materials
  • Tris‐buffered saline (TBS; appendix 2A), pH 7.2, 4°C
  • Brain tissue, fixed and sectioned (unit 1.1)
  • NAS blocking solution (for PAP method; see recipe)
  • NAS/avidin blocking solution (for ABC method; see recipe)
  • Monoclonal or polyclonal primary antibody against antigen of interest
  • Primary antibody diluent (see recipe)
  • Vectastain ABC peroxidase kit (for ABC method; Vector Labs) containing:
  •  Reagent A (avidin)
  •  Reagent B (biotinylated horseradish peroxidase)
  • Unlabeled secondary antibody (for PAP method; recognizing both primary antibody and PAP complex)
  • Biotin‐conjugated secondary antibody (for ABC method; Vector Labs)
  • Secondary antibody diluent (see recipe)
  • Peroxidase‐anti‐peroxidase (PAP) complex (for PAP method; from the same source species as the primary antibody; Sternberger Monoclonals)
  • PAP diluent: TBS ( appendix 2A) containing 2% NAS (for PAP method)
  • DAB substrate solution (see recipe; prepare immediately before use)
  • 0.1 M sodium nitrate
  • 10% (v/v) Triton X‐100
  • 0.1% (w/v) cresyl violet in 0.08% (v/v) acetic acid
  • 70%, 95%, and 100% ethanol
  • Histo‐Clear (National Diagnostics) or xylenes
  • Mounting medium: e.g., Permount (Fisher) or DPX (Electron Microscopy Sciences)
  • 6‐, 12‐, or 24‐well tissue culture plates
  • Glass transfer pipets with the ends heat‐sealed and bent to form a hook
  • Platform shaker
  • 0.22‐µm syringe filters (Millipore) and appropriate‐sized syringes
  • Mounting dish or large petri dish
  • Superfrost Plus microscope slides (Fisher)
  • Fine‐tipped paint brush
  • 60°C water bath
  • Slide rack
  • 24 × 60‐mm coverslips

Basic Protocol 2: Double‐Labeling Immunofluorescence and Quantitation of Confocal Images

  Materials
  • Tris‐buffered saline (TBS; appendix 2A), pH 7.2, 4°C
  • Brain tissue, fixed and sectioned (unit 1.1)
  • 3% (v/v) H 2O 2 (dilute 30% H 2O 2 in TBS)
  • NAS/avidin blocking solution (see recipe; if amplification is not required, avidin can be omitted)
  • Monoclonal and/or polyclonal primary antibody against antigen of interest
  • Primary antibody diluent (see recipe; if amplification is not required, biotin can be omitted)
  • Donkey anti‐species fluorescent conjugated secondary antibodies for multiple labeling (Jackson Immunoresearch): these antibodies have been preabsorbed to multiple species, resulting in minimal cross‐reactivity and minimal background staining; fluorescein, rhodamine‐red X and Cy5 (Cy5 can be used for triple labeling experiments) provide fluorophores with minimal overlap of emission/excitation spectra
  • Secondary antibody diluent (see recipe)
  • Donkey anti‐species biotin‐conjugated secondary antibodies for multiple labeling (Jackson Immunoresearch)
  • Vectastain ABC peroxidase kit (Vector Labs) containing:
    • Reagent A (avidin)
    • Reagent B (biotinylated horseradish peroxidase)
  • Tyramide signal amplification (TSA) fluorescence system kit (Perkin‐Elmer) including:
    • Tyramide‐fluorophore solution
    • Amplification diluent
  • Cupric sulfate solution (see recipe)
  • 10% (v/v) Triton X‐100
  • Vectashield mounting medium for fluorescence (Vector Labs)
  • Clear nail polish
  • 6‐, 12‐, or 24‐well tissue culture plates
  • Glass transfer pipets with the ends heat‐sealed and bent to form a hook
  • Platform shaker
  • Mounting dish or large petri dish
  • Superfrost Plus microscope slides (Fisher)
  • Fine‐tipped paint brush
  • 60°C water bath
  • Confocal laser scanning microscope system (unit 2.2), incorporating either an upright or inverted microscope
  • Metamorph Imaging System software (Universal Imaging Corp.) with colocalization module (other software may provide similar analyses)
  • Microsoft Excel software

Basic Protocol 3: Pre‐Embedding Immunohistochemistry for Electron Microscopy

  Materials
  • 0.1 M sodium phosphate buffer working solution, pH 7.4 (1× PB; see recipe for 2× stock)
  • 2% glutaraldehyde in 1× PB
  • 1% osmium tetroxide (see recipe)
  • 0.05 M sodium acetate, pH 7.3 (store up to several weeks at 4°C)
  • 2% uranyl acetate in 0.05 M sodium acetate, pH 7.3 (prepare fresh)
  • Propylene oxide (Electron Microscopy Sciences)
  • Epoxy resin (e.g., Epon from Ted Pella or Durcupan from Electron Microscopy Sciences)
  • Release‐agent‐coated microscope slides (see recipe)
  • Platform shaker
  • Glass transfer pipets
  • Shell vials or scintillation vials with stoppers
  • Wooden applicators
  • Resin stubs (see recipe)
  • 60°C oven
  • Additional reagents and equipment for immunohistochemical processing of tissue sections (as for light microsocopy; see protocol 1)
CAUTION: Osmium tetroxide is a highly reactive reagent. Wear appropriate protective gear such as gloves and use only in a chemical fume hood. Used osmium tetroxide should be stored in a specifically designated waste bottle and disposed of according to institutional procedures.
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Figures

Videos

Literature Cited

   Adams, J.C. 1981. Heavy metal intensification of DAB‐based HRP reaction product (letter). J. Histochem. Cytochem. 29:775.
   Berkeley, J.L., Gomeza, J., Wess, J., Hamilton, S.E., Nathanson, N.M., and Levey, A.I. 2001. M1 muscarinic acetylcholine receptors activate extracellular signal‐related kinase in CA1 pyrimidal neurons in mouse hippocampal slices. Mol. Cell Neurosci. 18:512‐524.
   Coligan, J.E., Kruisbeek, A.M., Margulies, D.H., Shevach, E.M., and Strober, W. (eds.) 1997. Current Protocols in Immunology. John Wiley & Sons, New York.
   Gilmor, M.L., Nash, N.R. Roghani, A., Edwards, R.H., Yi, H., Hersch, S.M., and Levey, A.I. 1996. Expression of the putative vesicular acetylcholine transporter in rat brain and localization in cholinergic synaptic vesicles. J. Neurosci. 16:2179‐2190.
   Levey, A.I., Kitt, C.A., Simonds, W.F., Price, D.L., and Brann, M.R. 1991. Identification and localization of muscarinic acetylcholine receptor proteins in brain with subtype‐specific antibodies. J. Neurosci. 11:3218‐3226.
   Levey, A.I., Hersch, S.M., Rye, D.B., Sunahara, R.K., Niznik, H.B., Kitt, C.A., Price, D.L., Maggio, R., Brann, M.R., and Ciliax, B.J. 1993. Localization of D1 and D2 dopamine receptors in rat, monkey, and human brain with subtype‐specific antibodies. Proc. Natl. Acad. Sci. U.S.A. 90:8861‐8865.
   Levey, A.I., Edmunds, S.M., Hersch, S.M., Wiley, R.G., and Heilman, C.J. 1995a. . Light and electron microscopic study of m2 muscarinic acetylcholine receptor in the basal forebrain of the rat. J. Comp. Neurol. 351:339‐356.
   Levey, A.I., Edmunds, S.M., Koliatsos, V., Wiley, R.G., and Heilman, C.J. 1995b. Expression of m1‐m4 muscarinic acetylcholine receptor proteins in rat hippocampus and regulation by cholinergic innervation. J. Neurosci. 15:4077‐4092.
   Schnell, S.A., Staines, W.A., and Wessendorf, M.W. 1999. Reduction of lipofuscin‐like autofluorescence in fluorescently labeled tissue. J. Histochem. Cytochem. 47:719‐730.
   Sternberger, L.A., Hardy, P.H., Cuculis, J.J., and Meyer, H.G. 1970. The unlabeled antibody enzyme method of immunohistochemistry. J. Histochem. Cytochem. 18:315‐333.
   Volpicelli, L.A., Lah, J.J. and Levey, A.I. 2001. Rab5 regulates targeting of the m4 subtype of muscarinic acetylcholine receptor to early endosomes and multivesicular bodies. J. Biol. Chem. 276:47590‐47598.
   Volpicelli, L.A., Lah, J.J., Fang, G., Goldenring, J.R. and Levey, A.I. 2002. Rab11a and the atypical myosin Vb direct transit of the M4 subtype of muscarinic acetylcholine receptor through the recycling pathway. J. Neurosci. 22:9776‐84.
   Volpicelli‐Daley, L.A., Duysen, E.G., Lockridge, O., and Levey, A. 2003a. Altered hippocampal and cortical muscarinic acetylcholine receptors in acetylcholinesterase deficient mice. Ann. Neurol. 53:788‐796.
   Volpicelli‐Daley, L.A. Hrabovska, A. Duysen, E.G., Ferguson, S.M., Blakely, R.D., Lockridge, O., and Levey, A. 2003b. Altered striatal function and muscarinic cholinergic receptors in acetylcholinesterase knockout mice. Mol. Pharmacol. In press.
   Zhang, W., Basile, A.S., Gomeza, J. Volpicelli, L.A., Levey, A.I., and Wess, J. 2002. Characterization of central inhibitory muscarinic autoreceptors by the use of muscarinic acetylcholine receptor knock‐out mice. J. Neurosci. 22:1709‐1717.
Key References
   Cuello, A.C. 1993. Immunohistochemistry II (IBRO Handbook Series: Methods in the Neurosciences). John Wiley & Sons, Chichester, U.K.
  An excellent resource on immunohistochemistry, dealing especially with immunoperoxidase and immunogold methods of pre‐ and post‐embedding electron‐microscopic immunohistochemistry, as well as with anterograde and retrograde tracing techniques.
   Dawes, C.J. 1971. Biological Techniques in Electron Microscopy (International Textbook Series). Barnes & Noble, New York.
  An excellent overview of electron microscopy including a brief historical synopsis, discussion of how an electron microscope works, several methods for tissue processing and thin‐sectioning, and methods for photographic techniques.
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