Eclipse e200 microscope

Animals were euthanized at 6 weeks of age and were transcardiac perfused with 4% paraformaldehyde (PFA); samples of liver were fixed in 4% PFA, dehydrated and processed to wax using standard procedures. The wax blocks were cut at 4-μm-thick sections, mounted on SuperFrost Plus slides (Thermo Scientific, Waltham, MA, USA) and subjected to hematoxylin and eosin (H&E) staining by standard methods. Images were taken on a Nikon eclipse E200 microscope with a Leica digital camera system.

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.

Tissue samples including macroscopic tumour, spleen, kidney, liver, and diaphragm were collected for histological and genetic analysis (RNAlater, QIAEN). Cell lines were generated from peritoneal ascitic fluid and macroscopic tumour when possible (see below). Tissues were fixed in 4% formaldehyde (Amber Scientific Pty Ltd., Perth, Western Australia) for 24–48 h, preserved in 70% ethanol prior to embedding in paraffin blocks (Surgipath Paraplast paraffin Leica Biosystems, Australia). Five micrometre (5 µm) sections were cut and stained with haematoxylin and eosin (H&E). Histopathological analysis was performed via bright field microscopy (Nikon Eclipse E200 microscope (Minato City, Tokyo, Japan), with selected sections scanned using Leica (Aperio) Scanscope Digital Slide Scanner. Ten (10) histological features were reviewed with each feature defined as representative of either benign or malignant disease. Additionally, the histological subtype of each mouse was also noted (epithelioid, sarcomatoid or biphasic).