The reproducibility of scrapie-associated fibril and PrPSc detection methods after long-term cold storage of natural ovine scrapie-affected brain tissue

A pool of grey matter (medulla/brain stem, cerebellum and frontal cerebral cortex) was prepared from the brains of 16 sheep with scrapie, diagnosed cl inically and by the demonstration of spongiform encephalopathy. Aliquots fr om the pool of tissue were finely chopped or homogenized and stored at +4 d egrees C or -70 degrees C, after undergoing one of several specific pre-tre atments (storage with or without protease inhibitors or: alternatively, wit h or without the cryoprotectant, dimethyl sulphoxide). At intervals over a period of 2 years, the stored extracts were examined by electron microscopy for the presence of scrapie-associated fibrils (SAFs) and by Western immun oblotting for the disease-specific abnormal protein PrPSc. Throughout the 2 -year period, SAFs and PrPSc were detected in the majority of all stored ti ssue extracts under all combinations of tissue preparation and pre-treatmen t. The combined detection rates for SAFs and PrPSc were 91% at +4 degrees C and 94% at -70 degrees C. There was no significant difference between the results obtained by the two detection methods and no specific combination o f preparation method and pre-treatment was superior to any other. Storage o f the samples at -70 degrees C appeared to give better results than storage at +4 degrees C, particularly with regard to fibril detection. For logisti cal reasons and ease of processing, and to avoid the effects of autolysis o n recognizable brain regions, long-term storage at -70 degrees C, without a ny pre-treatment, would appear to be the method of choice.