Lyso id red dye

Zeiss LSM 710 inverted confocal microscope was used to obtain all the confocal microscopy images. SMB and CAD cells were grown on cover slips in 24 well plates (at a concentration of 4 × 106). In general cells were fixed for 20 min in ice cold 4% PFA at 4 °C. Fixed cells were washed and permeabilised with 0.1% triton X100 for 10 min at 4 °C and blocked with 2% FCS for 30 min at RT before staining with primary antibodies. To observe PrP^Sc aggregates an additional step of treating the cells with 5M guanidine isothiocynate for 5–10 min at room temperature after permeabilising them with 0.5% tritonX-100 in PBS was employed. Live imaging for lysosomes was done using LYSO-ID red dye (Enzo life sciences, ENZ-51005-0100) (1:1000) by incubating the cells for 30 min at 37 °C. Proteostat dye (Enzo life sciences, ENZ-51035) was used to visualise the aggregates, cells were washed and fixed and permeabilised as above and incubated with Proteostat dye and Hoechst (1:100) and incubated in dark for 30 min at RT, and cells were later washed and mounted on glass slides.