Collected tissues were immediately processed into 1 × 1 mm pieces. Prefixation was performed in 2% paraformaldehyde (PFA) and 2.5% glutaraldehyde in 0.1 M phosphate buffer for 2 hours at 4°C. After osmication in 1% osmium tetroxide, the specimens were dehydrated and embedded. To obtain a wide field of view images using multiscale electron microscopy (MS‐EM), slices of resin‐embedded tissues were cut using an ultramicrotome (Leica EM‐UC6), placed on conductive, hydrophilic, coated glass slides, and stained with 1% toluidine blue. The images were digitalized using NanoZoomer software (NanoZoomer Digital Pathology, NDP view 2.2.1; Hamamatsu Photonics) and saved as digital slides. The same slice was stained with 1% uranyl acetate and lead and viewed on a scanning electron microscope (Hitachi SU8010). Tiling images were constructed with ImageJ software (National Institutes of Health). Image data were saved as virtual slides for MS‐EM. NanoZoomer software (Hamamatsu Photonics) was used for a wide field of view images.
Nanozoomer software
Manufactured by Hamamatsu Photonics
Sourced in Japan
The NanoZoomer software is a digital slide scanning and viewing system developed by Hamamatsu Photonics. It is designed to capture high-resolution images of microscope slides and manage the digital data. The software provides tools for slide scanning, image viewing, and basic image analysis functionalities.
Automatically generated - may contain errors
Lab products found in correlation
Slide scanner, by Hamamatsu Photonics (5 mentions)
Nanozoomer slide scanner, by Hamamatsu Photonics (1 mentions)
Em uc6, by Leica (1 mentions)
Su8010, by Hitachi (1 mentions)
β galactosidase reporter gene staining kit, by Merck Group (1 mentions)
Gram stain, by Merck Group (1 mentions)
Nanozoomer digital pathology system, by Hamamatsu Photonics (1 mentions)
α amylase, by Merck Group (1 mentions)
11 protocols using nanozoomer software
1
Multiscale Electron Microscopy Tissue Imaging
2
Quantifying Adipocytes in PWAT
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Female perirenal white adipose tissue (PWAT) was formalin-fixed and paraffin-embedded before sectioning and H&E staining. Sections were scanned with a Hamamatsu slide scanner (Children’s Research Institute Imaging Core, Medical College of Wisconsin) and .ndpi image files were analyzed for both cell counts and average area using Nanozoomer software (Hamamatsu). For each animal, six 0.25 mm2 areas were quantified for average number of adipocytes within the section (LH: n = 14; LH17LNa: n = 16) as well as average area of adipocytes within the section (LH: n = 15; LH17LNa: n = 16).
3
Quantifying Airway Inflammation via BALF Analysis
4
4NQO-Induced Oral Carcinogenesis
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Tongue tissues and lymph nodes were harvested at different time-points during 4NQO treatment. Ear skin was collected as a control for sequencing, before the start of the 4NQO treatment. WES was also performed on untreated tongues from C57BL/6 N mice following the same protocol (Supplementary Fig. 1 ) to provide germline DNA for the elimination of single-nucleotide polymorphisms. For frozen sections, tissues were embedded in OCT (optimal cutting temperature compound, VWR), sectioned and post-fixed in 4% paraformaldehyde/PBS pH 7.4 for 10 min before staining. For paraffin sections, tongue samples were fixed with 10% neutral buffered formalin overnight before paraffin embedding. The tissues were sectioned and stained with haematoxylin and eosin (H&E) by conventional methods. Images were acquired using a Hamamatsu slide scanner and analysed using NanoZoomer software (Hamamatsu).
5
Skin Keratinocyte Culture on Dermal Matrix
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Pre-confluent keratinocyte cultures (km) were disaggregated and transfected with SMART pool siRNAs or non-targeting control siRNAs, or with lentiviral shRNAs. 24 hr post-siRNA transfection or >7 days post-shRNA infection, keratinocytes were collected and reseeded on irradiated de-epidermised human dermis (Sen et al., 2010 (link)) in six-well Transwell plates with feeders and cultured at the air–liquid interface for 3 weeks. The cultures were then fixed in 10% neutral buffered formalin (overnight), paraffin embedded and sectioned for histological staining. 6 μm thick sections were labelled with haematoxylin and eosin or appropriate antibodies. H and E stained images were acquired with a Hamamatsu slide scanner and analysed using NanoZoomer software (Hamamatsu).
6
Quantifying Protein Expression Intensity
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All TMA slides were scanned at 200X magnification and scored visually using NanoZoomer software (Hamamatsu Photonics, Welwyn Garden City, UK). Both the staining intensity (negative, low, moderate and high) and percentage of staining were assessed, and a final H-score was calculated per sample. The H score were dichotomised into two main categories (high and low) using a cut-point value obtained using X-tile bioinformatics software [21 (link)].
Chi-squared (Χ2) test was used to study the association between the categorised data of P38/p-P38 and clinicopathological criteria. Kaplan–Meier was used to plot survival curves of OS and Log-Rank tests were used to estimate their significance. Cox multivariate analysis was performed using the log-rank test. A two-tailed p value < 0.05 was considered significant. Statistical analyses were performed using SPSS 21 statistical software (SPSS IBM Corp, Chicago, USA) and reported in line with REMARK guidelines [22 (link)].
Chi-squared (Χ2) test was used to study the association between the categorised data of P38/p-P38 and clinicopathological criteria. Kaplan–Meier was used to plot survival curves of OS and Log-Rank tests were used to estimate their significance. Cox multivariate analysis was performed using the log-rank test. A two-tailed p value < 0.05 was considered significant. Statistical analyses were performed using SPSS 21 statistical software (SPSS IBM Corp, Chicago, USA) and reported in line with REMARK guidelines [22 (link)].
7
Quantifying Adipocyte Size and Number
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Subcutaneous adipose tissue (iWAT) was fixed in 10% neutral buffered formalin for 24 h at 25 °C, embedded in paraffin, sectioned onto 5 µm sections, and hematoxylin and eosin (H&E) stained. The stained tissue was imaged using NanoZoomer software version 2.3.1 (Hamamatsu, Japan). Whole slide images were imported and analyzed using Visopharm VIS software version 2032.01 (Hørsholm, Denmark). Adipocyte size and number were quantified by thresholding for cell membranes; structures between 100 and 40,000 µm2 with a circularity index of 3.5 or less were considered adipocytes.
8
Histological Staining of Skin Samples
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Skin samples were fixed with 10% neutral buffered formalin overnight before paraffin embedding. The tissues were sectioned and stained with haematoxylin and eosin (H&E) by conventional methods (for more information on the antibodies, see supplementary material). Images of H&E‐stained sections were acquired using a Hamamatsu slide scanner and analysed using NanoZoomer software (Hamamatsu). For lacZ staining, a β‐galactosidase reporter gene staining kit (Sigma) was used, following the manufacturer's protocol (for information on the procedure used for oil red O staining of tail epidermal sheets, see supplementary material).
9
Tissue Preparation and Staining Protocol
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For frozen sections, tissues were embedded in OCT (optimal cutting temperature compound), sectioned and post-fixed in 4% paraformaldehyde/PBS pH 7.4 for 10 min before staining. X-gal staining on frozen sections was performed as described54 (link). The tissues were sectioned and stained with haematoxylin and eosin (H&E) and with Gram stain (Sigma) by conventional methods. Images were acquired using a Hamamatsu slide scanner and analysed using NanoZoomer software (Hamamatsu).
10
Histopathological Analysis of Chronic Otitis Media
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Tissue samples were fixed in 10% formaldehyde (Surgipath Europe) for 48 hours at room temperature and the skulls decalcified in Kristenson D.F.B agent (Pioneer Scientific) for 72 hours before wax embedding. Four µm H&E stained sections of the bullae and nasal passages were scanned using the NanoZoomer Digital Pathology system and morphometric measurements made with NanoZoomer software (Hamamatsu). Thickness of the ME mucosa was measured in a defined 1 mm area in the promontory region opposite the tympanic membrane. Average mucosal thickness was calculated using 5 measurements taken at a distance of 250 μm within this region. Both male and female mice were analysed and no sex-related differences were seen in the development of OM. H&E slides for sections of a total of 42 target tissues from Bpifa1−/− mice at 6 months of age were analysed for morphological abnormalities.
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