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IHC Detection PrP Intestine of Cattle Naturally Affected BSE

flounder

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Immunohistochemical Detection of Disease-Associated Prion Protein in the Intestine of Cattle Naturally Affected with Bovine Spongiform Encephalopathy by Using an Alkaline-Based Chemical Antigen Retrieval Method

Hiroyuki OKADA1), Yoshihumi IWAMARU1), Morikazu IMAMURA1), Kentaro MASUJIN1), Takashi YOKOYAMA1) and Shirou MOHRI1)

1) Prion Disease Research Center, National Institute of Animal Health

(Received 18-May-2010) (Accepted 17-Jun-2010)

ABSTRACT. An alkaline-based chemical antigen retrieval pretreatment step was used to enhance immunolabeling of disease-associated prion protein (PrPSc) in formalin-fixed and paraffin-embedded tissue sections from cattle naturally affected with bovine spongiform encephalopathy (BSE). The modified chemical method used in this study amplified the PrPSc signal by unmasking PrPSc compared with the normal cellular prion protein. In addition, this method reduced nonspecific background immunolabeling that resulted from the destruction of the residual normal cellular form of prion protein, and reduced the treatment time compared with the usual autoclave pretreatment step. Immunolabeled PrPSc was thereby clearly detected in the myenteric plexus of the ileum in naturally occurring BSE cattle.

KEY WORDS: antigen retrieval, BSE, cattle, immunohistochemistry, prion

http://www.jstage.jst.go.jp/article/jvms/advpub/0/advpub_1006220267/_article

This is, to the best of our knowledge, the first description of the localization of PrPSc within the colon of naturally affected BSE cattle. In the present study, PrPSc was observed in the myenteric plexus of the ileum and colon, but not in the duodenum and jejunum, using immunohistochemistry, western blot analysis, and mouse bioassay (Table 3). PrPSc has been detected in the myenteric plexus of the ileum from naturally affected BSE cattle [10, 12], chronic wasting disease in deer [22], and sheep scrapie [1], Based on the current BSE research, PrPSc is not found in gastrointestinal tissues other than the distal ileum [27]. BSE infectivity of the peripheral nervous system (PNS) was found in one clinical case [4]. PrPSc accumulation in the PNS of BSE-affected cattle during the clinical stages of infection may be the rule in BSE rather than the exception; it also may be the main mechanism rather than merely preceding the accumulation in the central nervous system (CNS) [14]. Results from western blotting and mouse bioassay indicated relatively low amounts of PrPSc deposits in the ileum and colon. 12 These data suggest that detectable PrPSc in the ileum and colon is probably caused by centrifugal spread from the CNS to the ENS within the peripheral nerves during the clinical stage of the disease. Therefore, to determine whether PrPSc was present in the ENS, the corresponding tissues should be subjected to western blot analysis by using the highly sensitive protocols described in this study or analyzed with a more sensitive mouse bioassay, or both.

In summary, chemical pretreatment with NaOH may provide a useful procedure for the detection of immunolabeled PrPSc and the suppression of the remaining PK-resistant PrPC without any nonspecific background immunostaining in FFPE tissue sections from BSE-affected cattle compared to the usual autoclave pretreatment step. In addition, pretreatment using NaOH at a higher temperature in the alkaline-based chemical antigen retrieval method clearly enhanced the strength of the immunolabeled PrPSc signal.

http://www.jstage.jst.go.jp/article/jvms/advpub/0/1006220267/_pdf



Monday, July 05, 2010

Immunohistochemical Detection of Disease-Associated Prion Protein in the Intestine of Cattle Naturally Affected with Bovine Spongiform Encephalopathy


http://bse-atypical.blogspot.com/2010/07/immunohistochemical-detection-of.html




tarball, aka flounder, alias TSS
 
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