Vectorshield hardset mounting medium

Mice were anaesthetised with 3% iso uorane (2l/min O 2 ) and transcardially perfused with 20 ml of icecold PBS, followed by 20 ml of ice-cold 4% PFA. Following xation, brains were immediately removed, and further xed overnight in 4% PFA at 4 C. Brains were processed in para n wax and sliced at 5 µM using a microtome. Following antigen retrieval, sections were washed in TBS + 0.1% triton X-100 and blocked for 2 hours at RT in TBS, 0.1% triton X-100, 10% goat serum and 5% BSA. Sections were incubated with an anti-PrP antibody (1:1000, ICSM35, D-Gen) to recognise preferentially PrP Sc over PrP C , an anti-GFAP (1:1000, ab134436, Abcam) or an anti-NeuN (1:1000, MAB377, Millipore). Sections were washed three times and incubated with Alexa Fluor uorescent secondary antibody for 1 hour at RT in blocking buffer. Following three washes, slices were mounted in Vectorshield hardset mounting medium with/without DAPI (Santa Cruz). For cell number analysis, pyramidal neurons were counted in hippocampal or cortical sections, normalised to area and data averaged from three brains per condition. All images were taken using a Zeiss LSM 510 META NLO microscope with ZEN 2009 software (Zeiss).