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.
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.