Scrapie material was obtained by endpoint titration of one passage of scrapie in ovine VRQ (tg338) transgenic mice. Two scrapie strains, 21K slow and CH1641, were further transmitted to sheep by intracerebral inoculation. Both strains were produced in Cheviot sheep with VRQ/VRQ genotype; the animals were age-matched and kept under the same feeding and environmental conditions. After the propagation of scrapie in sheep, strain typing was performed for each sheep brain isolate by back transmission in tg338 mice to make sure there was no strain divergence between the original and propagated prions.

Brain tissue was collected from euthanized sheep. A total of two isolates weighing 20 g was used for 21K slow strain, and three isolates weighing 10 g for CH1641 strain. Further on, using a glass tissue homogenizer grinder, 20% (w/v) brain tissue homogenates were prepared for each isolate in 1X phosphate-buffered saline (PBS, pH 7.4) containing protease inhibitor cocktail (cOmplete, EDTA-free Protease Inhibitor Cocktail, Roche) and 4% N-lauroylsarcosine sodium salt (sarkosyl, Sigma). Brain homogenates were centrifuged at 1,000 g for 10 minutes at room temperature in a bench centrifuge (Eppendorf), and 15 mL of aliquots of the supernatants (SNs) were transferred to clean 50 mL tubes (Falcon) and stored at -80°C until PrPSc purification. In the end, a total of 130 mL and 150 mL of 20% brain homogenate was used for 21K slow and CH1641 strain, respectively.

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