E0431

Five-µm-thick hepatic tissue sections were deparaffinized in xylene and rehydrated serially with alcohol and water, followed by microwave antigen retrieval for 20 min at 98 °C in 10 mM sodium citrate buffer (0.05% tween 20, pH 6.0). Endogenous peroxidase was blocked with fresh 0.3% hydrogen peroxide in phosphate buffered saline (PBS) for ten minutes at room temperature. Sections were blocked 2 h with protein block (DAKO, Belgium) in PBS containing 0.5% triton and were incubated overnight at 4 °C in the presence of a specific alpha-smooth muscle actin antibody (α-SMA, 1/200, ab5694, Abcam, UK). After being washed three times in PBS, sections were incubated with biotinylated secondary swine anti-rabbit antibody (E0431, DAKO, Belgium) for 1 h and with streptavidine-HRP (1/800, P0397, DAKO, Belgium) for 30 min. α-SMA was visualized using DAB (K3468, DAKO, Belgium) and sections were counterstained with hematoxylin. Sections were observed at 20X magnification Mirax Desk and Mirax viewer, Carl Zeiss MicroImaging, Germany).

Immunofluorescence analysis of BM sections was performed, as described by Herndler-Brandstetter et al. (8 (link)). Formalin-fixed, paraffin-embedded 4-µm BM sections were deparaffinized in xylene and re-hydratated in ethanol. The slides were boiled in 0.01 M citrate buffer (pH 6) for 16 min in the microwave for epitope retrieval and allowed to cool for about 1 h at room temperature. Slides were blocked with 3% skim milk in TBS/Tween for 20 min at room temperature. Rabbit anti-IL-15 (1:200; ab55276; Abcam) and mouse anti-CD8 (1:50; C8/144B; Dako) Abs were incubated overnight at 4°C. After washing, the slides were incubated for 1 h at 4°C with biotinylated swine anti-rabbit Ab (1:300; E0431; Dako) and a goat anti-mouse Alexa Fluor 546 Ab (1:300; A11018; Molecular Probes). Following washing steps with TBS/0.1% Tween, the BM sections were stained with a streptavidine-Alexa Fluor 488 Ab (1:500; S11223; Molecular Probes) for 30 min at 4°C. The stained slides were analyzed using confocal microscopy with an m-Radiance confocal scanning system (Bio-Rad) attached to a Zeiss Axiophot microscope (Carl Zeiss). For the quantitative analysis of CD8⁺ T cells in close contact with IL-15–producing cells in the BM, pictures from different areas of the BM sections were analyzed. In total, 800–1,000 CD8⁺ T cells were analyzed from each donor to calculate the percentage of contact with IL-15⁺ cells.

The fixed brains were embedded in paraffin, and subsequently cut in sections of 5 µm. After deparaffinization with xylene and rehydration through graded ethanol series, the slides were cooked for 20 min in citrate buffer for antigen retrieval. Slides were then stained overnight at 4°C with anti-amyloid-β antibody (1:1000; Abcam ab2539) followed by a 1 hr room temperature incubation with biotinylated secondary antibody (1:300; Dako E0431). Immunodetection was visualized using an Avidin-Biotin Complex kit (Vector Laboratories, UK), and sections were counterstained with haematoxylin before mounting. The slides were digitized with an automatic bright field microscope (Philips Ultra Fast Scanner, Philips, the Netherlands) and assessed by one examiner (LPM) for positivity for amyloid-β.