Detection of PrPScin brains of asymptomatic mice. A 10% brain homogenate from five (#1-#5) of six asymptomatic mice inoculated with 10–6 dilution of infected homogenate (Table 1) was prepared, and amplified by serial PMCA. Quadruplicate samples were analyzed after each round (R1–R4) of amplification by western blotting after digestion with proteinase K. One (#1 and #3), two (#5) and four (#2) of the quadruplicate samples were found to be positive for PrPSc after three or four rounds of amplification. No PrPSc signal was detected in #4 mouse. The eight lanes labeled “N” are samples in which only the PrPC substrate was treated in the same manner. Horizontal lines indicate the positions of molecular-weight markers corresponding to 37, 25, 20, and 15 kDa. nt: not tested.