Nanozoomer sq digital slide scanner

MAC-3⁺ immunohistochemical staining of mouse heart tissue was used to assess macrophage infiltration. Mouse heart tissue was washed with cold phosphate buffered saline, fixed with 4% paraformaldehyde for 6–8 h, and embedded in paraffin. Cardiac tissue sections (5 μmol thick) were stained with an antibody against the macrophage specific marker MAC-3 (BD Biosciences, rat derived, dilution 1:200). The sections were imaged with a NanoZoomer-SQ digital slide scanner (Hamamatsu Photonics, Shizuoka, Japan). To evaluate macrophage infiltration, 10 fields were randomly selected from each section, The percentage of MAC-3 positive staining (brown stained part) in the whole myocardial area was calculated by Image Pro Plus 6.0 image analysis software. Immunohistochemical positive staining (brown staining) was evaluated using GraphPad Prism software (version 8.0.1, GraphPad Software Inc., San Diego, CA, United States).

Hamamatsu NanoZoomer-SQ Digital slide scanner (C13140-01) with a x20 (NA 0.75) objective was used to obtain digital images of the slides. Slides were randomly blinded to the investigator. For each of the blinded slides, a representative 1 mm² area was selected employing NanoZoomer Digital Pathology viewer. The slide-image was cropped, containing the area of interest, and saved, as a TIF-file at × 20 resolution. The saved TIF-files were cropped, using ImageJ (Fiji) (44 (link)), into merely encompassing the 1 mm² area of interest.

A cardiac terminal blood withdrawal was performed at the time of sacrifice. Pancreas and tumors were extracted from mice at 1, 2, 3 and 4 weeks after orthotopic or heterotopic tumor implantation. Tissues were formalin fixed for 48 h prior to paraffin embedding. Sections were cut at 5 µm thickness. Representative sections were stained with Hematoxylin and Eosin (H&E) and Sirius Red to detect fibrosis. IHC staining for CD31 (1:200; #77699, Cell Signaling Technology, Danvers, MA, USA), alpha smooth muscle actin (αSMA) (1:100; #A5228, Sigma-Aldrich, St Louis, MO, USA) and cleaved caspase-3 (1:200; #9664, Cell Signaling Technology) was performed according to standard protocols. Images were captured with a Nikon Eclipse microscope equipped with an Insight CMOS 5.1 digital camera. Whole slides were scanned using a NanoZoomer-SQ Digital slide scanner (Hamamatsu Photonics K. K, Hamamatsu City, Japan). Sirius Red staining was used to measure CPA for tumor and pancreas samples (Polasek et al., 2012 (link); Fuchs et al., 2013 (link)). All image analysis was performed using ImageJ (NIH).

Mouse median liver lobes collected 46 h post sporozoite inoculation were fixed in 4% formaldehyde/PBS (Enzifarma) and paraffin embedded (Leica) in a transversal orientation. Stereology-sectioning into 3 μm thick slices was carried out using a Microtome (RM2145, Leica) and slides were stained with hematoxylin (Bio-optica) and eosin (Merck). Analysis of tissue sections was performed using a NanoZoomer-SQ Digital slide scanner (Hamamatsu Photonics) and included identification of intracellular parasite forms, hepatocellular damage and inflammatory infiltrates.

Organs were harvested, fixed (10% formalin), embedded in paraffin, sectioned (3-μm-thick sections), and stained with Hematoxylin and Eosin (H&E). Whole sections were analyzed, and images acquired with a Leica DMLB2 microscope (Leica) and NanoZoomer-SQ Digital slide scanner (Hamamatsu).