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Neuropathological Techniques to Investigate Central Nervous System Sections in Multiple Sclerosis

  • Jan Bauer
  • Hans LassmannEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1304)

Abstract

Immunohistochemical techniques (IHC) and in situ hybridization (ISH) are widely used techniques to study the expression of proteins and messenger RNAs in tissues and are extremely important to confirm and interpret biochemical and molecular results from the same tissues. Investigation of human brain by IHC and ISH therefore still plays an important role in the elucidation of pathogenetic mechanisms in diseases such as multiple sclerosis. In this review we describe the processing of human brain tissues as well as basic and advanced immunohistochemical staining and ISH techniques used for neuropathological analysis of such pathological brains.

Keywords

Immunohistochemistry In situ hybridization Neuropathology Multiple sclerosis Paraffin embedded Antigen retrieval 

Notes

Acknowledgments

We thank Ulrike Köck and Marianne Leißer for their technical assistance and their help with the writing of the various protocols.

References

  1. 1.
    Lassmann H (2007) Experimental models of multiple sclerosis. Rev Neurol 163:651–655PubMedCrossRefGoogle Scholar
  2. 2.
    Wekerle H (2008) Lessons from multiple sclerosis: models, concepts, observations. Ann Rheum Dis 67(Suppl 3):iii56–iii60PubMedCrossRefGoogle Scholar
  3. 3.
    Schuh C, Wimmer I, Hametner S, Haider L, Van Dam AM, Liblau RS et al (2014) Oxidative tissue injury in multiple sclerosis is only partly reflected in experimental disease models. Acta Neuropathol 128:247–266PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Fischer MT, Wimmer I, Hoftberger R, Gerlach S, Haider L, Zrzavy T et al (2013) Disease-specific molecular events in cortical multiple sclerosis lesions. Brain 136:1799–1815PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Bradl M, Lassmann H (2012) Microarray analysis on archival multiple sclerosis tissue: pathogenic authenticity outweighs technical obstacles. Neuropathology 32:463–466PubMedCrossRefGoogle Scholar
  6. 6.
    King G, Payne S, Walker F, Murray GI (1997) A highly sensitive detection method for immunohistochemistry using biotinylated tyramine. J Pathol 183:237–241PubMedCrossRefGoogle Scholar
  7. 7.
    Bauer J, Eiger CE, Hans VH, Schramm J, Urbach H, Lassmann H et al (2007) Astrocytes are a specific immunological target in Rasmussen’s encephalitis. Ann Neurol 62:67–80PubMedCrossRefGoogle Scholar
  8. 8.
    Hopman AH, Ramaekers FC, Speel EJ (1998) Rapid synthesis of biotin-, digoxigenin-, trinitrophenyl-, and fluorochrome-labeled tyramides and their application for In situ hybridization using CARD amplification. J Histochem Cytochem 46:771–777PubMedCrossRefGoogle Scholar
  9. 9.
    Bien CG, Bauer J, Deckwerth TL, Wiendl H, Deckert M, Wiestler OD et al (2002) Destruction of neurons by cytotoxic T cells: a new pathogenic mechanism in Rasmussen’s encephalitis. Ann Neurol 51:311–318PubMedCrossRefGoogle Scholar
  10. 10.
    Toth ZE, Mezey E (2007) Simultaneous visualization of multiple antigens with tyramide signal amplification using antibodies from the same species. J Histochem Cytochem 55:545–554PubMedCrossRefGoogle Scholar
  11. 11.
    Pardue ML, Gall JG (1969) Molecular hybridization of radioactive DNA to the DNA of cytological preparations. Proc Natl Acad Sci U S A 64:600–604PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Breitschopf H, Suchanek G, Gould RM, Colman DR, Lassmann H (1992) In situ hybridization with digoxigenin-labeled probes: sensitive and reliable detection method applied to myelinating rat brain. Acta Neuropathol 84:581–587PubMedCrossRefGoogle Scholar
  13. 13.
    Hart BA, Bauer J, Muller HJ, Melchers B, Nicolay K, Brok H et al (1998) Histopathological characterization of magnetic resonance imaging-detectable brain white matter lesions in a primate model of multiple sclerosis—a correlative study in the experimental autoimmune encephalomyelitis model in common marmosets (Callithrix jacchus). Am J Pathol 153:649–663PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Lucchinetti C, Bruck W, Parisi J, Scheithauer B, Rodriguez M, Lassmann H (2000) Heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelination. Ann Neurol 47:707–717PubMedCrossRefGoogle Scholar
  15. 15.
    Bauer J, Bradl M, Klein M, Leisser M, Deckwerth T, Wekerle H et al (2002) Endoplasmic reticulum stress in PLP-overexpressing transgenic rats: gray matter oligodendrocytes are more vulnerable than white matter oligodendrocytes. J Neuropathol Exp Neurol 61:12–22PubMedGoogle Scholar
  16. 16.
    Lucchinetti CF, Mandler RN, McGavern D, Bruck W, Gleich G, Ransohoff RM et al (2002) A role for humoral mechanisms in the pathogenesis of Devic’s neuromyelitis optica. Brain 125:1450–1461PubMedCrossRefGoogle Scholar
  17. 17.
    Kutzelnigg A, Lucchinetti CF, Stadelmann C, Bruck W, Rauschka H, Bergmann M et al (2005) Cortical demyelination and diffuse white matter injury in multiple sclerosis. Brain 128:2705–2712PubMedCrossRefGoogle Scholar
  18. 18.
    Lassmann H (2011) Review: the architecture of inflammatory demyelinating lesions: implications for studies on pathogenesis. Neuropathol Appl Neurobiol 37:698–710PubMedCrossRefGoogle Scholar
  19. 19.
    Hametner S, Wimmer I, Haider L, Pfeifenbring S, Bruck W, Lassmann H (2013) Iron and neurodegeneration in the multiple sclerosis brain. Ann Neurol 74:848–861PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Kutzelnigg A, Faber-Rod J, Bauer J, Lucchinetti C, Sorensen P, Laursen H et al (2007) Widespread demyelination in the cerebellar cortex in multiple sclerosis. Brain Pathol 17:38–44PubMedCrossRefGoogle Scholar
  21. 21.
    Kap YS, Bauer J, Driel N, Bleeker WK, Parren PW, Kooi EJ et al (2011) B-cell depletion attenuates white and gray matter pathology in marmoset experimental autoimmune encephalomyelitis. J Neuropathol Exp Neurol 70:992–1005PubMedCrossRefGoogle Scholar
  22. 22.
    Bien CG, Vincent A, Barnett MH, Becker AJ, Blümcke I, Graus F et al (2012) Immunopathology of autoantibody-associated encephalitides: clues for pathogenesis. Brain 135:1622–1638PubMedCrossRefGoogle Scholar
  23. 23.
    Van der Loos CM, Das PK, Van den Oord JJ, Houthoff HJ (1989) Multiple immunoenzyme staining techniques. Use of fluoresceinated, biotinylated and unlabelled monoclonal antibodies. J Immunol Methods 117:45–52PubMedCrossRefGoogle Scholar
  24. 24.
    Aboul-Enein F, Rauschka H, Kornek B, Stadelmann C, Stefferl A, Bruck W et al (2003) Preferential loss of myelin-associated glycoprotein reflects hypoxia-like white matter damage in stroke and inflammatory brain diseases. J Neuropathol Exp Neurol 62:25–33PubMedGoogle Scholar
  25. 25.
    Warford A, Akbar H, Riberio D (2014) Antigen retrieval, blocking, detection and visualisation systems in immunohistochemistry: a review and practical evaluation of tyramide and rolling circle amplification systems. Methods pii: S1046-2023(14)00094-2Google Scholar
  26. 26.
    Sargent PB (1994) Double-label immunofluorescence with the laser scanning confocal microscope using cyanine dyes. Neuroimage 1:288–295PubMedCrossRefGoogle Scholar
  27. 27.
    Berlier JE, Rothe A, Buller G, Bradford J, Gray DR, Filanoski BJ et al (2003) Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy dyes: fluorescence of the dyes and their bioconjugates. J Histochem Cytochem 51:1699–1712PubMedCrossRefGoogle Scholar
  28. 28.
    Marlatt MW, Bauer J, Aronica E, van Haastert ES, Hoozemans JJ, Joels M et al (2014) Proliferation in the Alzheimer hippocampus is due to microglia, not astroglia, and occurs at sites of amyloid deposition. Neural Plast 2014:693851PubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Neuroimmunology, Center for Brain ResearchMedical University of ViennaViennaAustria

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