Acta Neuropathologica

, Volume 87, Issue 1, pp 86–90 | Cite as

Hydrated autoclave pretreatment enhancement of prion protein immunoreactivity in formalin-fixed bovine spongiform encephalopathy-affected brain

  • M. Haritani
  • Y. I. Spencer
  • G. A. H. Wells
Regular Paper


The efficacy of three pretreatment techniques for the detection of prion protein (PrP) in formalin-fixed, paraffin-embedded bovine spongiform encephalopathy (BSE)-affected brain tissue were compared using automated image analysis. The most abundant immunostaining was in the form of particulate expression observed in sections pretreated with hydrated autoclaving for 30 min. Considerably less immunostaining occurred in sections pretreated with formic acid and no specific particulate immunostaining was detected in sections pretreated with hydrolytic autoclaving. Hydrated autoclaving pretreatment of sections prior to PrP immunolabelling gives visualisation of widespread sites of abnormal PrP deposition in the brain, allowing detailed study of the form and distribution of the protein in routinely fixed bovine central nervous system affected with BSE.

Key words

Immunohistochemistry Medulla oblongata Prion protein (PrP) Bovine spongiform encephalopathy Formalin-fixed tissue 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Bell JE, Ironside JW (1993) Neuropathology of spongiform encephalopathies in humans. Br Med Bull (in press)Google Scholar
  2. 2.
    Bradley R, Matthews D (1992) Sub-acute, transmissible spongiform encephalopathies: current concepts and future needs. Rev Sci Tech Off Int Epiz 11: 605–634Google Scholar
  3. 3.
    Bruce ME, McBride PA, Farquhar CF (1989) Precise targeting of the pathology of the sialoglycoprotein, PrP, and vacuolar degeneration in mouse scrapie. Neurosci Lett 102: 1–6PubMedGoogle Scholar
  4. 4.
    Dawson M, Mansley LM, Hunter AR, Stack MJ, Scott AC (1987) Comparison of scrapie associated fibril detection and histology in diagnosis of natural sheep scrapie. Vet Rec 121: 591Google Scholar
  5. 5.
    Doi-Yi R, Kitamoto T, Tateishi J (1991) Immunoreactivity of cerebral amyloidosis is enhanced by protein denaturing treatments. Acta Neuropathol 82: 260–265PubMedGoogle Scholar
  6. 6.
    Farquhar CF, Somerville RA, Ritchie LA (1989) Post-mortem immunodiagnosis of scrapie and bovine spongiform encephalopathy. J Virol Methods 24: 215–221PubMedGoogle Scholar
  7. 7.
    Gibson PH, Somerville RA, Fraser H, Foster JD, Kimberlin RH (1987) Scrapie associated fibrils in the diagnosis of scrapie in sheep. Vet Rec 120: 125–127PubMedGoogle Scholar
  8. 8.
    Hope J, Mason J (1991) The scrapie fibril protein and its cellular isoform. Curr Top Microbiol Immunol 172: 57–74PubMedGoogle Scholar
  9. 9.
    Hope J, Reekie LJD, Hunter N, Multhaup G, Beyreuther K, White H, Scott AC, Stack MJ, Dawson M, Wells GAH (1988) Fibrils from brains of cows with new cattle disease contain scrapie-associated protein. Nature 336: 390–392PubMedGoogle Scholar
  10. 10.
    Ironside JW, McCardle L, Hayward PAR, Bell JE (1993) Ubiquitin immunocytochemistry in human spongiform encephalopathies. Neuropathol Appl Neurobiol 19: 134–140PubMedGoogle Scholar
  11. 11.
    Jeffrey M, Halliday WG, Goodsir CM (1992) A morphometric and immunohistochemical study of the vestibular nuclear complex in bovine spongiform encephalopathy. Acta Neuropathol 84: 651–657PubMedGoogle Scholar
  12. 12.
    Kimberlin RH (1992) Bovine spongiform encephalopathy. Rev Sci Tech Off Int Epiz 11: 347–390Google Scholar
  13. 13.
    Kitamoto T, Ogomori K, Tateishi J, Prusiner SB (1987) Formic acid pretreatment enhances immunostaining of cerebral and systemic amyloids. Lab Invest 57: 230–236PubMedGoogle Scholar
  14. 14.
    Kitamoto T, Muramoto T, Mohri S, Doh-ura K, Tateishi J (1991) Abnormal isoform of prion protein accumulates in follicular dendritic cells in mice with Creutzfeldt-Jakob disease. J Virol 65: 6292–6295PubMedGoogle Scholar
  15. 15.
    Kitamoto T, Doh-ura T, Muramoto K, Miyazono M, Tateishi J (1992) The primary structure of the prion protein influences the distribution of abnormal prion protein in the central nervous system. Am J Pathol 141: 271–277PubMedGoogle Scholar
  16. 16.
    McBride PA, Bruce ME, Fraser H (1988) Immunostaining of scrapie cerebral amyloid plaques with antisera raised to scrapie-associated fibrils (SAF). Neuropathol Appl Neurobiol 14: 325–336PubMedGoogle Scholar
  17. 17.
    Miyazono M, Kitamoto T, Doh-ura K, Iwaki T, Tateishi J (1992) Creutzfeldt-Jakob disease with codon 129 polymorphism (valine): a comparative study of patients with codon 102 point mutation or without mutations. Acta Neuropathol 84: 349–354PubMedGoogle Scholar
  18. 18.
    Prusiner SB (1982) Novel proteinaceous infectious particles cause scrapie. Science 216: 136–144PubMedGoogle Scholar
  19. 19.
    Rubenstein R, Merz PA, Kascsak RJ, Carp RI, Scalici CL, Fama CL, Wisniewski HM (1987) Detection of scrapie-associated fibrils (SAF) and SAF proteins from scrapie-affected sheep. J Infect Dis 156: 36–42PubMedGoogle Scholar
  20. 20.
    Scott AC, Done SH, Venables C, Dawson M (1987) Detection of scrapie-associated fibrils as an aid to the diagnosis of natural sheep scrapie. Vet Rec 120: 280–281PubMedGoogle Scholar
  21. 21.
    Scott AC, Wells GAH, Stack MJ, White H, Dawson M (1990) Bovine spongiform encephalopathy: detection and quantitation of bibrils, fibril protein (PrP) and vacuolation in brain. Vet Microbiol 23: 295–304PubMedGoogle Scholar
  22. 22.
    Scott AC, Wells GAH, Chaplin MJ, Dawson M (1992) Bovine spongiform encephalopathy: detection of fibrils in the central nervous system is not affected by autolysis. Res Vet Sci 52: 332–336PubMedGoogle Scholar
  23. 23.
    Serban D, Taraboulos A, DeArmond SJ, Prusiner SB (1990) Rapid detection of Creutzfeldt-Jakob disease and scrapie prion proteins. Neurology 40: 110–117PubMedGoogle Scholar
  24. 24.
    Shin RW, Iwaki T, Kitamoto T, Tateishi J (1991) Hydrated autoclave pretreatment enhances TAU immunoreactivity in formalin-fixed normal and Alzheimer's disease brain tissues. Lab Invest 64: 693–702PubMedGoogle Scholar
  25. 25.
    Stack MJ, Scott AC, Done SH, Dawson M (1991) Natural scrapie: detection of fibrils in extracts from the central nervous system of sheep. Vet Rec 128: 539–540PubMedGoogle Scholar
  26. 26.
    Sternberger LA, Hardy PH Jr, Cuculis JJ, Meyer HG (1970) The unlabelled antibody enzyme method of immunohistochemistry preparation and properties of soluble antigen-antibody complex horseradish-D peroxidase anti-horseradish-D peroxidase and its use in the identification of spirochetes. J Histochem Cytochem 18: 315–333PubMedGoogle Scholar
  27. 27.
    Taraboulos A, Serban D, Prusiner SB (1990) Scrapie prion protein accumulates in the cytoplasm of persistently infected cultured cells. J Cell Biol 110: 2117–2132PubMedGoogle Scholar
  28. 28.
    Wells GAH, Scott AC, Johnson CT, Gunning RF, Hancock RD, Jeffrey M, Dawson M, Bradley R (1987) A novel progressive spongiform encephalopathy in cattle. Vet Rec 121: 419–420PubMedGoogle Scholar
  29. 29.
    Wells GAH, Hancock RD, Cooley WA, Richards MS, Higgins RJ, David GP (1989) Bovine spongiform encephalopathy: diagnostic significance of vacuolar changes in selected nuclei of the medulla oblongata. Vet Rec 125: 521–524PubMedGoogle Scholar
  30. 30.
    Wells GAH, Wilesmith JW, McGill IS (1991) Bovine spongiform encephalopathy: a neuropathological perspective. Brain Pathol 1: 69–78PubMedGoogle Scholar
  31. 31.
    Wells GAH, Hawkins SAC, Hadlow WJ, Spencer YI (1992) The discovery of bovine spongiform encephalopathy and observations on the vacuolar changes. In: Prusiner SB, Collinge J, Powell J, Anderton B (eds) Prion disease of humans and animals. Ellis-Horwood, Chichester, pp 256–274Google Scholar
  32. 32.
    Wilesmith JW, Wells GAH, Cranwell MP, Ryan JBM (1988) Bovine spongiform encephalopathy: epidemiological studies. Vet Rec 123: 638–644PubMedGoogle Scholar
  33. 33.
    Wilesmith JW, Ryan JBM, Hueston WD (1992) Bovine spongiform encephalopathy: case-control studies of calf feeding practices and meat and bone meal inclusion in proprietary concentrates. Res Vet Sci 52: 325–331PubMedGoogle Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • M. Haritani
    • 1
  • Y. I. Spencer
    • 1
  • G. A. H. Wells
    • 1
  1. 1.Department of PathologyCentral Veterinary LaboratoryAddlestoneUK

Personalised recommendations