Antigen retrieval using sodium hydroxide for prion immunohistochemistry in bovine spongiform encephalopathy and scrapie.
Formalin-fixed and paraffin wax-embedded (FFPE) tissue sections are usually used for histopathological and immunohistochemical analyses in prion diseases in animals and man. However, formalin fixation cross-links proteins, reducing disease-associated prion protein (PrP(Sc)) immunolabelling. To detect PrP(Sc) in animals naturally affected with bovine spongiform encephalopathy (BSE) and scrapie, we applied minimal pretreatment with sodium hydroxide (NaOH). This simple pretreatment, combined with enzymatic digestion using proteinase K (PK), was equally effective in the detection of PrP(Sc) in FFPE tissue, and superior in terms of speed, compared with the usual autoclaving method. The most effective results, without any section loss, were obtained with 10 μg/ml PK in phosphate buffered saline containing 0.1% Triton-X at room temperature for 10 min and 150 mM NaOH at 60 °C for 10 min. By this simple procedure, PrP(Sc) was visualized in the brain of animals with BSE and scrapie using a range of anti-PrP primary antibodies.
Prion Disease Research Team, Prion Disease Research Center, National Institute of Animal Health, 3-1-5 Kan-nondai, Tsukuba, Ibaraki 305-0856, Japan. okadahi@affrc.go.jp
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Okada
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Sato
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Sakurai
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Endo
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Yokoyama
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Mohri
D000818:Animals; D000941:Antigens / Q000737:chemistry*; D002417:Cattle; D016643:Encephalopathy, Bovine Spongiform / Q000276:immunology / Q000378:metabolism / Q000473:pathology*; D007150:Immunohistochemistry / Q000662:veterinary; D008526:Medulla Oblongata / Q000378:metabolism / Q000473:pathology; D018620:PrPSc Proteins / Q000032:analysis / Q000378:metabolism*; D012608:Scrapie / Q000378:metabolism / Q000473:pathology*; D012756:Sheep; D012972:Sodium Hydroxide / Q000737:chemistry*
D000941:Antigens; D018620:PrPSc Proteins; D012972:Sodium Hydroxide