Comparison of biochemical extraction techniques for the detection of scrapie-associated fibrils in the central nervous system of sheep naturally affected with scrapie.
Standardized samples of brain material from four sheep naturally affected with scrapie and from four healthy control sheep were subjected to six different extraction techniques used for the detection of scrapie-associated fibrils by negative-contrast transmission electron microscopy. The six methods were compared in respect of fibril yield and clarity of ultrastructure. The simplest method consisting of a single N-lauroylsarcosine detergent extraction and differential centrifugation, followed by proteinase K enzyme digestion, gave the best overall results. The use of proteinase and nuclease inhibitors made no apparent difference to the yield or ultrastructural clarity of fibrils. Density gradient centrifugation appeared to reduce tungstate stain penetration and often obscured the ultrastructural clarity. The results suggested that the preferred technique could be improved by the use of a double homogenization stage at the beginning of the procedure and by adding an ultrasonic disintegration step to resuspend the final pellet prior to tungstate staining.
Stack
M J
MJ
Veterinary Laboratories Agency, Central Veterinary Laboratory, Weybridge, Surrey, UK.
Aldrich
A M
AM
Kitching
A D
AD
Scott
A C
AC
D000818:Animals; D002493:Central Nervous System Diseases / Q000473:pathology*; D005260:Female; D016874:Neurofibrillary Tangles / Q000473:pathology*; D016900:Neurofilament Proteins / Q000302:isolation & purification* / Q000648:ultrastructure; D012608:Scrapie / Q000473:pathology*; D012756:Sheep
D016900:Neurofilament Proteins