Anti prp antibody 6d11

Brain sections treated with 0.3% Triton-X100 for 30 min and blocked with 5% BSA for 1 h at RT. The sections were incubated with primary antibodies, including anti-AQP1 (1:50), anti-AQP4 antibody (1:100), anti-AQP9 antibody (1:100), anti-PrP antibody (6D11, 1:300, Santa Cruz, Sc-58,581), anti-GFAP antibody (1:300, CST, #3670), at 4°C overnight. Subsequently, brain sections were incubated with Alexa Fluor® 488-conjugated goat anti-rabbit antibody (1:200, Thermo, A-11,008) or/and Alexa Fluor® 568-conjugated goat anti-mouse antibody (1:200, Thermo, A-11,004) at RT (brain sections at 37°C) for 1 h. After stained with 1 μg/mL 4ʹ6-diamidino-2-phenylindole (DAPI, Beyotime, China) for 30 min, the slices were mounted with Permount and viewed using Operetta (Perkinelmer) or Olympus FV1000 confocal microscopy.

The proteins in exosome samples were separated by 15% SDS-PAGE and electroblotted onto a nitrocellulose membrane using a semi-dry blotting system (Bio-Rad). Membranes were blocked at room temperature (RT) for 1 h with 5 % (w/v) nonfat milk powder in 1× Tris-buffered saline containing 0.1% Tween 20 (TBST) and then probed at 4°C overnight with primary antibodies, including anti-PrP antibody (6D11, SantaCruz, sc-58581), anti-β-actin antibody (Subrray Biotechnology, sr-25113), ALIX antibody (Abcam, ab275377), TSG101 antibody (Abcam, ab125011), and calnexin antibody (Abcam, ab22595). After washing with TBST, the blots were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (Jackson ImmunoResearch Labs, 115-035-003 and 111-035-003) at RT for 2 h. The blots were developed using an enhanced chemiluminescence system (ECL, PerkinElmer, NEL103E001EA) and visualized on autoradiography films (General Electric). Images were captured using a ChemiDoc™ XRS+ Imager (Bio-Rad). To detect the presence of proteinase K (PK)-resistant PrP^Sc, the exosome samples were digested with PK at a final concentration of 25 μg/ml at 37°C for 60 min prior to Western blotting. The PK digestion was terminated by heating the samples with loading buffer at 100°C for 10 min.