The histological features of the lesions were assessed in the same groups of mice described in Section 2.2.8.1 . For this purpose, at the end of those experiments, animals were sacrificed with CO2 narcosis, skin wounds were excised with a wide margin of normal skin (approx. 2 mm) and processed for routine histology. After fixation, trimming was performed longitudinally, in the direction of the hair flow and centered on the wound. Samples were embedded in paraffin, sectioned at 4 µm, and stained with hematoxylin and eosin (H&E). Histopathological analysis was performed by a pathologist blinded to experimental groups using a Nanozoomer SQ slide scanner and NDP.view2 software (Hamamatsu Photonics, Hamamatsu, Japan). Standard criteria were used to stage the lesion and to perform semi-quantitative analysis of the inflammatory cell infiltration [20 ].
Nanozoomer sq slide scanner
Manufactured by Hamamatsu Photonics
Sourced in Japan
The NanoZoomer SQ is a high-performance slide scanner designed for digital pathology applications. It captures high-resolution images of microscope slides with a maximum scan area of 15 mm x 15 mm and a resolution of up to 0.23 microns per pixel. The scanner utilizes a linear image sensor and a motorized stage to efficiently digitize entire slide samples.
Automatically generated - may contain errors
Lab products found in correlation
Axioskop microscope, by Zeiss (2 mentions)
Cal ex 2, by Thermo Fisher Scientific (2 mentions)
Axiocam camera, by Zeiss (2 mentions)
Streptavidin qdot 655, by Thermo Fisher Scientific (2 mentions)
Vectastain abc ap, by Vector Laboratories (2 mentions)
Mouse monoclonal antibody to α smooth muscle cell actin, by Merck Group (2 mentions)
Biotinylated griffonia simplifolia lectin 1, by Vector Laboratories (2 mentions)
Ndp view2, by Hamamatsu Photonics (1 mentions)
Fv1000 ix81, by Olympus (1 mentions)
Neo mount, by Merck Group (1 mentions)
Mayer s hemalum, by Merck Group (1 mentions)
6 protocols using nanozoomer sq slide scanner
1
Histological Assessment of Skin Wound Lesions
2
Histological Analysis of Mouse Brain
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After experiments (12 days' treatment in PD experiments), mice were sacrificed. And tissues were extracted and fixed with 4% paraformaldehyde, followed by dehydrate treatment and embedding in paraffin for histological, immunohistochemical, and immunofluorescence analysis. Following cutting and dewaxing, brain sections were initially washed in PBS for five minutes and then incubated in 3% methol-H2O2 for 15 minutes. After washing with PBS for three times, section were applied with high fire power in microwave oven for 4 minutes and low heat for 20 minutes and cooled to room temperature. Subsequently, they were incubated with primary antibodies at 4 °C overnight, then brain sections were subjected to secondary antibodies (Alexa Fluor® 488 conjugation for immunofluorescence) with 0.05% tween 20 for one hour at room temperature. After 3, 3-diaminobenzidine (DAB) coloration and hematoxylin counterstaining, the sections were counted after being hydrated again. All the pathological sections were scanned in their entirety with a NanoZoomer-SQ slide scanner (Hamamatsu, Japan). IHC analysis was conducted with the assistance of Image J software. The sections of immunofluorescence were observed by confocal microscopy (FV1000-IX81, Olympus, Japan).
3
Histological Analysis of Ischemic Limbs
4
Histological Analysis of Yolk Sac Fry
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Single yolk sac fry were fixed in 15 ml of freshly made 4% paraformaldehyde in PBS (pH 7.4) for 48 h with very gentle rotation at FRT and stored at 4°C until processing. After fixation, fry were carefully deyolked and cut in segments along the anteroposterior axis using an industrial razor blade. Body segments were infiltrated stepwise overnight from 80% ethanol to paraffin using a TP1020 Semi-enclosed Benchtop Tissue Processor. The segments were laid horizontally with the same orientation along the dorsoventral axis and aligned at their anterior ends in a preliminary paraffin block, before the embedding in a final paraffin block that exposed the anterior end of all segments for sectioning. Ten 4 µm sections were obtained per fish per body segment. Mucosubstances were stained with Alcian Blue/PAS and nuclei with Mayer’s hemalum (101646, 101647, and 109249, respectively; Merck). Slides were mounted using NeoMount (109016, Merck). Images were acquired using a NanoZoomer SQ slide scanner (Hamamatsu) at 40X. NanoZoomer digital pathology for SQ 1.0.5 software (Hamamatsu) was used for image acquisition and export, as well as analyses including cell count and epithelial length measurement.
5
Quantitative Immunohistochemical Analysis of Melanoma
6
Histological Analysis of Ischemic Limbs
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Histological analysis was performed on both the ischemic and non-ischemic limbs at day 21 post hind limb ischemia surgery. Mice were euthanized and the tissue was perfused with saline followed by 10% buffered formalin for fixation. The bone was then demineralized in a formic acid based solution (Cal-Ex II, Fisher Scientific) for 48 hours before processing and paraffin embedding. Sections (5 μm thick) were immunostained for vascular smooth muscle cells or endothelial cells. Enzyme treatment was performed in proteinase K (Biolabs 2ug/ml) prior to incubation with primary antibodies. For smooth muscle cells, the sections were stained using a mouse monoclonal antibody to α-smooth muscle cell actin (Sigma) as the primary antibody detected using the avidin-biotin-alkaline phosphatase method (Vectastain ABC-AP, Vector Laboratories) with a hematoxylin counter-stain. Images of the entire section were acquired used the Hamamatsu NanoZoomer SQ slide scanner. To visualize endothelial cells slides were stained with biotinylated Lectin antibody (Biotinylated Griffonia simplifolia Lectin 1, Vector lab), followed by incubation with Streptavidin Qdot 655 (Invitrogen). Images were acquired using the 20X Plan-Neo air objective on a Zeiss Axioskop microscope equipped with an AxioCam camera. ImageJ software (NIH) was used to count the number of vessels for analysis.
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