Multiplex Immunostaining for Neurodegenerative Markers

Immunohistochemistry was performed on 6 μm thick sections, with appropriate antigen retrieval steps. The primary antibodies (Supplementary Table 2) were used to recognize: (i) epitopes of pTau-—AT8, AT100, CP13, PHF1, pThr181, pSer356 and pSer396, and phosphorylated and unphosphorylated tau with Tau-2; (ii) microglial markers^{23 (link),24 (link)}—Iba1, CD68, HLA-DR, CD64, CD32a and CD16; (iii) astrocyte markers—GFAP, EAAT2, glutamine synthetase (GS), ALDH1L1; and (iv) T lymphocyte markers CD4 and CD8. Incubation with secondary biotinylated antibodies (Vector Laboratories) was visualized by avidin-biotin-peroxidase complex (Vectastain Elite, Vector Laboratories) and chromogenic reaction with 3,3′-diaminobenzidine (Vector Laboratories). Slides were counterstained with haematoxylin and coverslipped with Expert XTF Mounting Media (CellPath). Due to the large number of slides, staining was carried out in three batches. Each batch included all conditions and brain banks. One set of slides was stained with Haematoxylin and Eosin (H&E) to examine the cortical tissue integrity.