Lung samples were fixed in 10% buffered formalin (Thermo Fisher). Gross pathology images were acquired using a Power Shot G9 camera (Canon). For detailed histological analysis, lung lobes were embedded in paraffin, sectioned, and stained with H&E (Pulmonary Morphology Core, Washington University). Mtb bacilli were stained with Ziehl-Neelsen stain (Pulmonary Morphology Core). Images were acquired using NanoZoomer 2.0-HT System (Hamamatsu) or an Eclipse E400 camera (Nikon).
Nanozoomer 2.0 ht system
Manufactured by Hamamatsu Photonics
Sourced in Japan, Belgium
The NanoZoomer 2.0-HT system is a high-throughput digital slide scanning system designed for pathology and research applications. It captures high-resolution digital images of microscope slides, enabling efficient digitization and archiving of specimen samples.
Automatically generated - may contain errors
Lab products found in correlation
Matlab software r2013a, by MathWorks (2 mentions)
Ndp viewing software, by Hamamatsu Photonics (2 mentions)
Histostain sp kit, by Thermo Fisher Scientific (2 mentions)
Biotinylated universal secondary antibody, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (2 mentions)
Powershot g9 camera, by Canon (1 mentions)
Rat anti mouse cd45, by BD (1 mentions)
Axio scan z1, by Zeiss (1 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Dab solution, by Merck Group (1 mentions)
Abc solution, by Vector Laboratories (1 mentions)
Cytoseal 60, by Thermo Fisher Scientific (1 mentions)
Thunder imager 3d assay, by Leica (1 mentions)
Rabbit anti fibrinogen antibody, by Abcam (1 mentions)
Vectastain elite abc kit solution, by Vector Laboratories (1 mentions)
Biotinylated goat anti rabbit antibody, by Bio-Rad (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
Visiomorph software, by Visiopharm (1 mentions)
Matlab 8.3 r2014a software, by MathWorks (1 mentions)
29 protocols using nanozoomer 2.0 ht system
1
Lung Histopathology of Tuberculosis
2
Immunohistochemical Characterization of Bone Inflammation
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Tissues were fixed in 10% formalin for paraffin embedding. Tibias were decalcified in 14% EDTA prior to paraffin embedding. 5µm sections were stained by immunohistochemistry (IHC) with rat anti-mouse CD45 (BD Pharmingen #550539), CD3 (Dako #A0452), B220 (BD Pharmingen #550286). Flag-tagged HBZ expression was detected following citrate antigen retrieval with the anti-flag antibody (Sigma #F1804, 5ug/ml) and detected using the HistoMouse-Plus Broad Spectrum (AEC) kit (Life Technologies, catalog #849541). Tibias were stained with the tartrate-resistant acid phosphatase (TRAP) (Sigma-Aldrich) to visualize osteoclasts. The number and surface area of TRAP+ cells/ bone surface area were quantified at 5X magnification for WT (n = 7) and HBZ (n = 7) mice. Images were acquired using the NanoZoomer 2.0-HT System (Hamamatsu Photonics) or the Zeiss Axio Scan Z1.
3
Granulocyte Morphology Characterization
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Granulocytes (NDNs and LDNs) were classified as immature or mature according to their nuclear segmentation. Nuclei displaying >2 nuclear lobes were considered as mature, those with ≤2 lobes were classified as immature granulocytes (Fig. 2A ). At least 400 granulocytes were evaluated by an assessor blinded to sample origin.
All slides were then digitized at 200× magnification with the NanoZoomer 2.0-HT system (Hamamatsu Photonics, SZK, Japan). The diameter of each type of granulocyte was measured using ImageJ (http://rsb.info.nih.gov/ij/ ) with cells approximated as circles. The evaluator was blinded to slide identification and measurements were over 200 randomly selected granulocytes.
All slides were then digitized at 200× magnification with the NanoZoomer 2.0-HT system (Hamamatsu Photonics, SZK, Japan). The diameter of each type of granulocyte was measured using ImageJ (
4
Volumetric Analysis of Hippocampus
5
Immunohistochemical Staining Protocol for Brain Sections
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Sections were stained in a free-floating format in a 12-well plate. After extensive washing with TBS, sections were blocked with 0.3% hydrogen peroxide for 10 min. After washing, sections were blocked for 30 min in 3% milk in TBS and 0.25% Triton X-100 (TBS-X). The respective biotinylated primary antibody was diluted in 3% milk in TBS-X, and the sections were incubated overnight at 4°C. After washing, sections were incubated with ABC solution (VectaStain) according to the manufacturer’s instructions, followed by another three wash cycles. Sections were developed in DAB solution (Sigma-Aldrich), washed, and mounted on slides. After drying overnight, sections were dehydrated in increasing ethanol concentrations followed by xylene. Slides were coverslipped with Cytoseal 60 (Thermo Fisher Scientific) and scanned on a NanoZoomer 2.0-HT system (Hamamatsu Photonics). Images were further processed and quantified with the use of ImageJ/Fiji software version 2.0.0. All areas were quantified in at least four sections (300 µm apart from each other) per mouse.
6
Quantifying Lung Tumor Metastasis
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Histologic sections of paraffin-embedded lung and thymus tissues (5 μm) were stained with hematoxylin and eosin (HE) and imaged using the digital slide scanner NanoZoomer 2.0-HT system (0.46 μm/pixel (20X) scanning resolution) (Hamamatsu Photonics, Hamamatsu City, Japan). Metastatic dissemination to lungs was evaluated on five HE-stained sections (separated each other by 50 μm) per mouse lung by measuring tumor area in lungs and reporting it to the total area of lung tissue analyzed. Quantification of lung tumor area was performed as described previously [48 (link)].
7
Quantification of Lung Inflammation
8
Murine AD-like Histopathology Analysis
9
Multimodal Histological Imaging
10
Quantifying Tumor Vascular Density and Perfusion
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Immunostained sections were scanned using the digital slide scanner NanoZoomer 2.0-HT system at 0.46 μm/pixel (20X) scanning resolution (Hamamatsu, Mont- Saint-Guibert, Belgium) and images were registered in the RGB (red, green, blue) color space. Necrotic and stromal areas were eliminated using the hybrid human-computer approach described previously (35 (link)). For blood vessel and functional blood vessels quantification within tumor regions of interest, image processing and measurements were performed using the toolbox of image analysis of the MATLAB (R2013a) software (Mathworks, Inc.) according to the algorithms as previously described elsewhere (36 (link)). Importantly, binary images resulting from the image processing were systematically compared visually with the corresponding original images and occasionally, the threshold was adapted manually if necessary. The results are expressed as density defined as the measured area occupied by vessels divided by the total area of the corresponding tumor regions of interest or for perfusion as the area of perfused vessels divided by the total area of vessels.
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