Tissuetek glove mounting media

Sections were stained using a Ventana Benchmark XT (Ventana). Deparaffinised sections were incubated for 30–60 min in CC1 solution (Ventana) for antigen retrieval. Primary antibodies were diluted in 5% goat serum (Dianova), 45% Tris-buffered saline pH 7.6 (TBS) and 0, 1% Triton X-100 in antibody diluent solution (Zytomed). Sections were then incubated with primary antibody for 1 h (see supplemental list). Anti-mouse histofine Simple Stain MAX PO Universal immunoperoxidase polymer (Nichirei Biosciences) were used as secondary antibody. Detection of secondary antibodies was performed with an ultraview universal DAB detection kit from Ventana with appropriate counterstaining and sections were cover-slipped using TissueTek glove mounting media (Sakura Finetek). To allow comparability for each analysis, sections of Tg(PrPΔ214–229) mice and controls were stained in one machine run.
For PrP^Sc staining, samples were treated for 30 min in Peroxidase-Blocking buffer (0, 3% H₂O₂) and 30 min in 98% formic acid. After washing with water, samples were autoclaved for 5 min at 121 °C in citrate buffer pH 6. The protocol was then followed as described above with the additional step of PK digestion followed by incubation of SAF84 antibody.

Processing of samples, including cutting and staining of paraffin sections with HE, was performed as published [39 (link)]. For immunohistochemistry, all sections were stained using the Ventana Benchmark XT machine (Ventana, Tuscon, Arizona, USA). Deparaffinised sections were boiled for 30–60 min in CC1 solution (Ventana, Tuscon, Arizona, USA) for antigen retrieval. Primary antibodies were diluted in 5% goat serum (Dianova), 45% Tris-buffered saline pH 7.6 (TBS) and 0.1% Triton X-100 in antibody diluent solution (Zytomed, Berlin, Germany). Sections were then incubated with primary antibody POM1 (1:100), Iba1 (Dako, 1:2.000) or GFAP (Dako, 1:400) for one hour. Anti-mouse or anti-rabbit histofine Simple Stain MAX PO Universal immunoperoxidase polymer (Nichirei Biosciences) was used as secondary antibody. Detection of secondary antibodies was performed with an ultraview universal DAB detection kit from Ventana with appropriate counterstaining and sections were cover-slipped using TissueTek glove mounting media (Sakura Finetek).
The neuropathological assessment (n = 3 or n = 4 depending on the genotype) was conducted by three independent investigators in a blinded fashion by grading the samples 1 (mild) to 3 (severe) depending on the staining intensity or spongiosis.