The following kits (Abcam) were used as per the manufacturer’s instructions: Senescence Detection Kit (SA-βGal) and Fontana-Masson (melanin). H&E staining was performed on de-paraffinized and rehydrated sections using hematoxylin and eosin (Sigma-Aldrich) and mounted in Entellan mounting medium. Cell infiltration was scored as: 0 = not detected, 1 = mild, 2 = moderate, 3 = severe. Cartilage staining was performed with toluidine blue (1%, 10 min) (Sigma-Aldrich). Cartilage integrity (loss of sulfated proteoglycans staining) was scored as: 0 = normal, 1 = mild loss, 2 = moderate loss, 3 = total loss of staining. Images were acquired at ×20 magnification on a Nanozoomer S210 Slide Scanner (Hamamatsu). Exposure and sharpness were kept constant in all images for comparison.
Nanozoomer s210 slide scanner
Manufactured by Hamamatsu Photonics
Sourced in Japan
The NanoZoomer S210 is a high-resolution digital slide scanner designed for the digitization of glass microscope slides. It captures detailed images of specimens with a maximum resolution of 0.23 micrometers per pixel. The device is capable of scanning slides up to 32 mm x 86 mm in size.
Automatically generated - may contain errors
Lab products found in correlation
Ndp view2 viewing software, by Hamamatsu Photonics (2 mentions)
Ndp view2, by Hamamatsu Photonics (2 mentions)
Nod scid il2yr female mice, by Jackson ImmunoResearch (2 mentions)
Cytation 7 instrument, by Agilent Technologies (2 mentions)
Toluidine blue, by Merck Group (1 mentions)
Hematoxylin and eosin, by Merck Group (1 mentions)
Ndp viewer software, by Hamamatsu Photonics (1 mentions)
Observer z1, by Zeiss (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Direct red 80, by Merck Group (1 mentions)
Slide scanner, by Leica (1 mentions)
Picrosirius red, by Merck Group (1 mentions)
Anti mlh1 clone g168 15, by BD (1 mentions)
12 protocols using nanozoomer s210 slide scanner
1
Histological Evaluation of Senescence and Melanin
2
X-gal Staining of Cryosectioned Tissue
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Due to the limited penetration of the X-gal substrate in larger tissues, X-gal staining was also performed directly on cryosections for time points over E13.5, and for all adult and neonatal MI hearts. Dissected hearts were fixed for 1–3 h, depending on size, in 4% PFA in PBS at 4 °C, then incubated overnight in 30% sucrose at 4 °C. They were then washed in a 50/50 mix of 30% sucrose/OCT Embedding Medium (Thermo Scientific), followed by a minimum of two washes in OCT before mounting over dry ice and storing at −80 °C.
Cryosections were cut at a thickness of 15 μm, and allowed to thaw at room temperature before washing in PBS to remove the OCT embedding medium. Sections were incubated in Fix solution (4% PFA, 2 mM MgCl2, 5 mM EGTA in PBS) for 10 min at room temperature, before further washes in PBS. They were then incubated in Cryosection Staining solution (2 mM MgCl2, 0.02% Nonidet P40, 0.01% sodium deoxycholate, 5 mM K4Fe(CN)6, 5 mM K3Fe(CN)6, and 1 mg/ml X-gal in PBS) in a humidified chamber at room temperature for 1 h to overnight, depending on staining intensity.
After staining, sections were washed in PBS, fixed in 4% PFA in PBS for 15 min, counter-stained with Nuclear Fast Red and imaged using a NanoZoomer S210 slide scanner with NDP.view2 viewing software (Hamamatsu).
Cryosections were cut at a thickness of 15 μm, and allowed to thaw at room temperature before washing in PBS to remove the OCT embedding medium. Sections were incubated in Fix solution (4% PFA, 2 mM MgCl2, 5 mM EGTA in PBS) for 10 min at room temperature, before further washes in PBS. They were then incubated in Cryosection Staining solution (2 mM MgCl2, 0.02% Nonidet P40, 0.01% sodium deoxycholate, 5 mM K4Fe(CN)6, 5 mM K3Fe(CN)6, and 1 mg/ml X-gal in PBS) in a humidified chamber at room temperature for 1 h to overnight, depending on staining intensity.
After staining, sections were washed in PBS, fixed in 4% PFA in PBS for 15 min, counter-stained with Nuclear Fast Red and imaged using a NanoZoomer S210 slide scanner with NDP.view2 viewing software (Hamamatsu).
3
Visualizing Tissue Matrisome Composition
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Serial sections of the HGS mouse tumors, along with normal omenta were stained by immunohistochemistry for all 6 ECM molecules and scanned with Hamamatsu NanoZoomer S210 Slide Scanner. Digital images from the serial sections were obtained using the NPD.view 2.7.25 software under the same magnification. The ImageJ (ImageJ 1.48v) image processing and analysis program (NIH, Bethesda, MD) with color threshold and color deconvolution plug-ins was used for the generation of tissue matrisome heatmaps. Briefly, images were aligned and color deconvoluted and all six isolated DAB images were subsequently converted to single color images and compiled to a Z stack (AVG stack), selecting average intensity. A tissue matrix heatmap was generated by applying an edited Royal filter to the AVG stack, so that each color corresponds to either the absence (black) or the presence in situ of one (dark blue), two (cyan), three (green), four (yellow), five (orange) or six (red) ECM molecules. Finally, a Calibration bar was applied to the AVG stack with all seven colors and the heatmap image was flattened before export.
4
Spatial Profiling of Tumor Microenvironment
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Sections were cut from diagnostic formalin-fixed-paraffin-embedded blocks sampled at resection. H&E slides pre- and post-LCM and post-CyCIF were scanned using the NanoZoomer S210 slide scanner (Hamamatsu, Welwyn Garden City, UK). Representative sections were selected for annotations, which were drawn on pre-LCM H&E slides as first choice in most cases. Annotations included:
Annotations were made using the NDPViewer software (Hamamatsu, Welwyn Garden City, UK). The tumour-normal interface was expanded by 500μm on either side to identify the invasive margin. Images with annotations were analysed using a digital cell classifier which uses deep learning methodology modelled on a spatially constrained neural network architecture29 (link). The model first detects cells via predicted location of cell nuclei, then classifies them as: normal epithelial, cancer epithelial, fibroblast, lymphocyte, neutrophil, macrophage, endothelial. Absolute counts are calculated for each annotated region. For each annotation, number of cells of a given type per epithelial cell was identified by dividing total number of cells of that type by total number of epithelial cells for that annotation.
In addition to the above, we used a further classifier35 (link) to class all tumour-associated lymphocytes as infiltrating (ITL) or adjacent (ATL) or distant (DTL) based on proximity to tumour cells.
Annotations were made using the NDPViewer software (Hamamatsu, Welwyn Garden City, UK). The tumour-normal interface was expanded by 500μm on either side to identify the invasive margin. Images with annotations were analysed using a digital cell classifier which uses deep learning methodology modelled on a spatially constrained neural network architecture29 (link). The model first detects cells via predicted location of cell nuclei, then classifies them as: normal epithelial, cancer epithelial, fibroblast, lymphocyte, neutrophil, macrophage, endothelial. Absolute counts are calculated for each annotated region. For each annotation, number of cells of a given type per epithelial cell was identified by dividing total number of cells of that type by total number of epithelial cells for that annotation.
In addition to the above, we used a further classifier35 (link) to class all tumour-associated lymphocytes as infiltrating (ITL) or adjacent (ATL) or distant (DTL) based on proximity to tumour cells.
5
Automated Microscopy Imaging Protocol
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Bright-field and fluorescence images were taken on a Zeiss inverted microscope (Observer.Z1) using Axiovision software (Zeiss). Mosaic pictures were automatically reconstructed from individual 10x (brightfield) or 20x (fluorescence) tiles. In some cases, whole slide imaging (WSI) was performed using a Nanozoomer S210 slide scanner (Hamamatsu, Japan).
6
Quantifying Lung Metastasis in Mice
7
Breast Tumor Mast Cell and Collagen Analysis
8
Pancreatic Cancer P2RY2 Expression
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Formalin-fixed paraffin-mbedded (FFPE) sections (n=3) of PDAC with stroma and normal adjacent tissue were obtained from the Barts Pancreas Tissue Bank (Project 2021/02/QM/RG/E/FFPE). Sections were stained using the human P2RY2 probe (853761, ACD) and the RNAscope 2.5 HD Assay-RED (ACD) following the manufacturer’s instructions. Slides were imaged by NanoZoomer S210 slide scanner (Hamamatsu).
9
Automated Quantification of Immunohistochemistry
10
Immunohistochemical Staining of FFPE Tissue Sections
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FFPE tissue sections (2–3 µm) were used for immunohistochemical staining [47 (link),48 (link)]. Briefly, sections were deparaffinized and rehydrated and subsequently stained according to standard protocols. The following primary antibodies were used: anti-CD3 (clone PS1, dilution 1:100, Abcam, Cambridge, UK); anti-MLH1 (clone G168-15, dilution 1:300, BD Pharmingen, Heidelberg, Germany); and anti-MSH2 (clone FE11, dilution 1:100, Calbiochem, Darmstadt, Germany). As a secondary antibody, the biotinylated anti-mouse/anti-rabbit antibody (Vector Laboratories) was used at 1:100 dilution. Staining was visualized using the Vectastatin elite ABC detection system (Vector Laboratories, Burlingame, CA, USA) and 3,3′-diaminobenzidine (Dako North America Inc., Carpinteria, CA, USA) as a chromogen. For counterstaining hematoxylin was used. Stained sections were scanned using NanoZoomer S210 slide scanner (Hamamatsu, Hamamatsu, Japan) and viewed using NDP.view2 Viewing Software (Hamamatsu). Four random 0.25 mm2 square regions were drawn in the tumor tissue and positive cells in each region were counted using the QuPath Software, giving mean values per 0.25 mm2.
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