Vectastain elite

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.

Brain sections obtained from the rostrocaudal level of the midstriatum were rinsed for 20 min in 0.3% H₂O₂ in 70% methanol/0.1 M phosphate-buffered saline (PBS), immersed in 0.1 M PBS containing 5% bovine serum albumin (BSA) (05470; Sigma-Aldrich, Darmstadt, Germany), and incubated for 1 h in biotinylated anti-mouse IgG (1:100; Santa Cruz, Heidelberg, Germany), followed by diaminobenzidine (DAB) tetrahydrochloride (D5905; Sigma-Aldrich) staining with an avidin-biotin complex peroxidase kit (Vectastain Elite; Vector Labs, Burlingame, CA, USA) [5 (link)]. IgG extravasation was analyzed by measuring the area of IgG leakage.

Kidney sections (3 μm thick) stained with periodic acid-Schiff (PAS) were used to evaluate glomerular lesions, such as glomerular cell proliferation, expansion of the mesangial matrix and segmental glomerulosclerosis. The severity of glomerular injury was determined according to a method described previously [21 , 49 (link)]. In brief, the severity of injury for each glomerulus was scored from 0 to 4: 0, no lesion; 1, <25% involvement of the glomerulus; 2, 25-50% involvement; 3, 50-75% involvement; and 4, >75% involvement. Fifty glomeruli were analyzed per kidney section. A glomerular sclerosis score (GSS) per animal was calculated by multiplying each severity score (0-4+) with the percentage of glomeruli displaying the same degree of injury and by summing these scores. The above histological analysis was performed in a blind manner to avoid bias. Immunohistochemical stains were performed on formalin-fixed, paraffin-embedded 3-μm sections. Primary antibodies against the following proteins were used: LC3 (1:1000), Nephrin (1:200), and Podocin (1:200). Staining was visualized using horseradish peroxidase-coupled secondary antibodies (Vectastain elite, Vector Labs). All immunohistochemical analyses were repeated at least three times, and representative images are shown.

Following rinses in hydrogen peroxide (2% in PB, 15 min) and Triton X-100 (2% in PB, 30 min), sections were incubated in a purified rabbit antiserum against GFP that also recognizes eYFP (1:500; EXBIO, Prague, Czech Republic) + 2% Triton X-100 + 3% normal goat serum (NGS) + 1% bovine serum albumin (BSA) in PB (16 h, 20°C). After several rinses in PB, sections were incubated in biotinylated goat anti-rabbit IgG (1:100; Sigma–Aldrich, St. Louis, MO, USA) + 2% Triton X-100 + 3% NGS + 1% BSA in PB (2 h, 20°C). Following new rinses in PB, the sections were incubated in avidin-biotin-peroxidase complex solution (1:100; Vectastain Elite, Vector Laboratories, Burlingame, CA, USA) + 2% Triton X-100 (4°C, 16 h). Peroxidase activity was visualized using a glucose oxidase-DAB-nickel protocol (Shu et al., 1988 (link)). Sections, were lightly counterstained with Thionin for cytoarchitectonic reference, defattened, dehydrated and coverslippped with DePeX.