PCLSs were fixed in 4% buffered formalin overnight and stored at 4 °C in 70% ethanol. Fixed PCLSs were embedded in paraffin and sectioned 4 μm thick. Tissue morphology and fibrosis were assessed through hematoxylin and eosin (H&E) staining (Hematoxyline: Cat. 4085.9002, Klinipath, Duiven, The Netherlands; Eosin: Cat. HT110232, Sigma-Aldrich) and Picrosirius Red (PSR) staining (ab150681, Abcam) according to the standard histological procedure. Stained tissue sections were scanned using a Nanozoomer Digital Pathology Scanner (NDP Scan U10074-01, Hamamatsu Photonics K.K., Shizuoka, Japan) [10 (link),13 ]. ImageJ was used to quantify the stained areas. The histopathologic percentages of necrosis and fat vesicles were determined by an experienced pathologist (MH).
Nanozoomer digital pathology scanner ndp scan u10074 01
Manufactured by Hamamatsu Photonics
Sourced in Japan
The Nanozoomer Digital Pathology Scanner (NDP Scan U10074-01) is a high-resolution digital pathology scanning system produced by Hamamatsu Photonics. The core function of this device is to capture digital images of prepared tissue samples on microscope slides.
Automatically generated - may contain errors
Lab products found in correlation
Ab150681, by Abcam (1 mentions)
Aperio imagescope viewer software, by Leica (1 mentions)
Anti cd3 polyclonal, by Agilent Technologies (1 mentions)
Goat anti rabbit igg hrp, by Agilent Technologies (1 mentions)
Rabbit anti mouse igg hrp, by Agilent Technologies (1 mentions)
Tissue tec oct compound, by Sakura Finetek (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
3 amino 9 ethylcarbazole, by Agilent Technologies (1 mentions)
4 protocols using nanozoomer digital pathology scanner ndp scan u10074 01
1
Histological Evaluation of Paraffin-Embedded Tissue Sections
2
Quantifying Macrophage Markers in Aortic Layers
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All slides were scanned and digitally stored using the Nanozoomer Digital Pathology Scanner (NDP Scan U10074-01; Hamamatsu Photonics K.K., Hamamatsu, Japan), and the Aperio ImageScope Viewer software (Leica Biosystems, Wetzlar, Germany) was used for quantification of the expression of each marker in arteries. A semiquantitative score was used for quantification as follows: 0 (absence of expression), 1 (≤ 1%), 2 (2-20%), 3 (20-50%) and 4 (> 50%) according to the frequency of positive cells in ×10 magnification fields [21] throughout the whole artery by two raters (A.W.S.S. and J.P.S.) who were blinded for the patient data. The mean score of each rater was calculated by summing the quantification of each cell marker in all fields of the aorta and by dividing for the total number of fields. Then, the mean score from both raters was calculated and used for statistical analysis. Seven aorta specimens presenting the best tissue integrity and clear distinction between layers throughout the arteries were chosen for the quantification of macrophage markers in each layer (i.e. intima, media and adventitia).
3
Detailed Kidney Pathology Analysis in Autoimmune Mice
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Kidney tissue samples from 30 week-old female IL-17RA KO lpr and B6/lpr mice were frozen in Tissue-Tec O.C.T. Compound (Sakura Finetek Europe B.V) and stored at −80 °C or embedded in formalin. Two µm sections of formalin-fixed, paraffin-embedded kidney tissues were cut and were routinely stained with haematoxylin or eosin (H&E) and periodic acid- Schiff (PAS) for evaluation of kidney pathology. Complement C3 and IgG staining was performed on 5 µm frozen kidney sections with 1 µg/ml rabbit anti-C3 antibody (Thermoscientific) followed by goat-anti-rabbit IgG-HRP (Dako). For IgG staining rabbit anti-mouse IgG-HRP (Dako) was used. Peroxidase activity was detected with DAB and sections were counterstained with Mayer’s hematoxylin. All sections were scored digitally after examination using a Nanozoomer Digital Pathology Scanner (NDP Scan U10074-01, Hamamatsu Photonics K.K., Japan) and quantified ((number of positive pixels* 0.5) + number of strong positive pixels/total pixels) with software of ImageScope Viewer (V11.2.0.780 Aperio, e-Pathology Solution, CA, USA).
HMGB1 and CD3 staining was performed on 2 µm paraffin sections using polyclonal anti-HMGB1 (Abcam, Cambridge, UK) and polyclonal anti-CD3 (Dakocytomation).
HMGB1 and CD3 staining was performed on 2 µm paraffin sections using polyclonal anti-HMGB1 (Abcam, Cambridge, UK) and polyclonal anti-CD3 (Dakocytomation).
4
Immunohistochemical Staining of IST1 in Formalin-Fixed Tissues
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Formalin-fixed, paraffin-embedded tissues were cut into sections of 3 µm. The sections were deparaffinized and rehydrated, followed by antigen retrieval with tris-EDTA buffer (pH 9) for 15 min in a microwave. Tissues were incubated with primary anti-human IST1 (66989-1-Ig, 1:1,000 dilution; Proteintech) for 1 h at room temperature (RT), followed by endogenous peroxidase blocking with 3% H2O2 for 30 min. The tissues were subsequently incubated with EnVision anti-mouse peroxidase conjugated secondary antibody (DAKO), 3-amino-9-ethylcarbazole (DAKO) for peroxidase activity detection, and finally hematoxylin (Merck) as a counterstain. All slides were scanned using a Nanozoomer Digital Pathology Scanner (NDP Scan U 10074-01; Hamamatsu Photonics). Images were analyzed using FIJI-ImageJ (Schindelin et al., 2012 (link)).
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