Dmd108 microscope

A protocol based on the Wachstein/Meisel lead phosphate method was used [8 (link),11 (link),44 (link),45 ]. The sections were washed twice with 50 mM Tris-maleate buffer pH 7.4 and pre-incubated for 30 min at RT with 50 mM Tris-maleate buffer pH 7.4 containing 2 mM MgCl₂ and 0.25 mM sucrose. The enzymatic reaction was carried out by incubating tissue sections for 1 h at 37 °C with 50 mM Tris-maleate buffer pH 7.4 supplemented with 0.25 mM sucrose, 2 mM MgCl₂, 5 mM MnCl₂, 3 % Dextran, 2 mM Pb(NO₃)₂, and 2 mM CaCl₂. All experiments were performed in the presence of 2.5 mM levamisole, as an inhibitor of alkaline phosphatase (AP) activity, and in the presence of 1 mM AMP, ADP, ATP, or TPP as a substrate. TPP is a false substrate, which can be cleaved by the pyrophosphatase activity of E-NPPs. Control assays were performed in the absence of nucleotide. For E-NTPDase inhibition experiments, 1 mM POM 1 was added to pre-incubation and enzymatic reaction buffers. For CD73 inhibition experiments, 1 mM α, β-meADP was added to pre-incubation and enzymatic reaction buffers. The reaction was revealed by incubation with 1% (NH₄)₂S (v/v) for exactly 1 min. Nuclei were counterstained with haematoxylin. Samples were mounted with aqueous mounting medium (FluoromountTM, Sigma-Aldrich), observed under a light Nikon Eclipse E200 microscope, and photographed under a light Leica DMD 108 microscope.

Liver samples for immunohistochemical analyses were selected from the tissue archives of the Institute of Pathology, Ludwig Maximilian University of Munich, Germany. Liver samples originated from patients with clinical indication for liver biopsy. Tissue sections were stained with anti-ITGB3 (Ventana) on an automated slide staining platform from Ventana, according to manufacturer’s instructions. For analysis, representative brightfield images were taken on a Leica DMD 108 microscope at the indicated magnifications. ITGB3-positive infiltration spots at inflammation sites were counted at 40× magnification.