Immunofluorescence Detection of PrPSc Aggregates in Astrocytes

For immunofluorescence-based detection of PrP^Sc aggregates in astrocytes, 1 × 10⁵ cells of prion-infected (22L, RML, and ME7) astrocytes were seeded in 12-well culture plate. Once 60–70% confluent, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature, followed by quenching of autofluorescence (with 50 mm NH₄Cl, 20 mm glycine) for 10 min at room temperature. The cells were permeablized with PBS containing 5% FBS and 0.3% Triton X-100 for 30 min followed by denaturation of PrP^C and epitope retrieval of PrP^Sc by incubation with 6 m guanidine HCl for 10 min at room temperature as described previously (44 (link), 45 (link)). The cells were incubated with anti-PrP mAb 4H11 (diluted at 1:100 in PBS containing 5% FBS and 0.3% Triton X-100) overnight at 4 °C. Alexa Fluor 488 goat anti-mouse secondary antibody (Jackson Immunoresearch) was used (at 1:200 in PBS containing 5% FBS and 0.3% Triton X-100) for 1 h at room temperature to visualize immunofluorescence. 4′,6′-Diamino-2-phenylindole (1:5000 in PBS) was used as a nuclear stain. Glial fibrillary acidic protein rabbit pAb was purchased from DAKO (Z0334). All images were captured under 63× oil lens at the same acquisition settings from a Zeiss LSM 700 confocal microscope.