Stack M J, Aldrich A M, Kitching A D, Scott A C
Veterinary Laboratories Agency, Central Veterinary Laboratory, Weybridge, Surrey, UK.
J Comp Pathol. 1996 Aug;115(2):175-84. doi: 10.1016/s0021-9975(96)80039-9.
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.
从四只自然感染羊瘙痒病的绵羊以及四只健康对照绵羊身上获取标准化的脑材料样本，采用六种不同的提取技术，通过负染透射电子显微镜检测羊瘙痒病相关纤维。对这六种方法在纤维产量和超微结构清晰度方面进行了比较。最简单的方法是先进行单次月桂酰肌氨酸去污剂提取和差速离心，然后进行蛋白酶K酶消化，总体效果最佳。使用蛋白酶和核酸酶抑制剂对纤维产量或超微结构清晰度没有明显影响。密度梯度离心似乎会降低钨酸盐染色的穿透力，且常常会模糊超微结构的清晰度。结果表明，在该程序开始时使用双重匀浆步骤，并在钨酸盐染色前增加超声破碎步骤以重悬最终沉淀，可改进首选技术。
