Anti mouse or anti rabbit secondary antibody

Postmortem brain tissue donation was approved and provided for this project by the Queen Square Brain Bank, University College London. Paraffin blocks from representative areas of the brain were available for histological and immunohistochemical investigation. In brief, 8-mm-thick sections of paraffin embedded tissue blocks were deparaffinised and rehydrated through graded alcohols. Sections were placed in 0.3% H₂O₂/methanol to block endogenous peroxidase activity. Antigen retrieval was undertaken to unmasked antigen epitopes and was achieved either by pressure cooking in citrate buffer (pH 6.0) for 10 min, incubation either in 99% formic acid for 10 min or in proteinase K solution (Dako) for 10 min. The sections were washed with tris-buffered saline and incubated in 10% non-fat milk solution to prevent non-specific antibody binding. The appropriate primary antibody was applied (see online supplementary table S4), followed by an antimouse or antirabbit secondary antibody (Dako, Ely, UK) at 1/200 dilution, as appropriate. Subsequently, avidinbiotin complex Elite kit (Vector, Peterborough, UK) was applied and colour was developed by diaminobenzidine/H₂O₂. Sections were finally counterstained with Mayer’s haematoxylin. Routine H&E histological staining was also carried out.