NR AXMI
AU Finlayson,J.; Hamilton,S.; Eaton,S.L.; Steele,P.J.; Dagleish,M.P.; Jeffrey,M.; Gonzalez,L.; Chianini,F.
TI Characterisation of Milk Cells by Immunohistochemistry and Western Blot
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Pathology and Pathogenesis P03.81
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Poster
AB Immune cells are believed to be involved in the pathogenesis of natural scrapie in sheep. Abnormal PrP (PrPsc) could be present in circulating immune cells of animals infected with transmissible spongiform encephalopathy (TSE) and they could be shed during inflammatory diseases such as mastitis. Most cases of mastitis occur in the first month of lactation, and as a consequence the number of immune cells present in milk increases. PrPsc has been shown previously to accumulate in lymphoid follicles close to the milk ducts and PrPc has been found in milk. Described here is a method to collect cells from milk to be used for Western Blot (WB) or to be labelled by immunohistochemistry (IHC). One hundred millilitres (mls) of sheep milk was collected for up to 3 days after lambing from animals of various genotypes from a natural scrapie infected flock kept at Moredun. On each day the 100mls of milk was divided into five ml. aliquots and mixed with 40 mls cold PBS and centrifuged at 800g for 10 minutes at 4°. The cream and supernatant were carefully removed and the pelleted cells resuspended with cold PBS. The cells were washed several times using centrifugation and cold PBS and divided equally into three aliquots. Two of the aliquots of cells were resuspended in 5% gelatine and allowed to solidify into plugs. The third aliquot of cells was resuspended in PBS then frozen at -20° for WB. The gelatine plugs of cells were fixed in either Zinc Salts fixative or 10% buffered formalin for 24 hours before being processed overnight then embedded in wax. Sections were cut and stained with Haematoxylin and Eosin showing that the cells present were morphologically well preserved. A panel of monoclonal antibodies (CD3, CD79, CD68) was used to characterise the cell subsets by IHC. PrPc was demonstrated in the prepared milk cells by WB using P4 antibody.
AD J. Finlayson, S. Hamilton, S.L. Eaton, P.J. Steele, M.P. Dagleish, F. Chianini, Moredun Research Institute, UK; M. Jeffrey, L. González, Veterinary Laboratory Agency, Lasswade, UK
SP englisch
PO Schottland