For histopathologic analysis, formalin-fixed liver and ileum samples were paraffin-embedded, sectioned (5 μm thickness), and stained with hematoxylin and eosin. The sections were randomly numbered prior to reading and observed by an experienced pathologist. Images were acquired using a NanoZoomer 2.0-RS scanner (Hamamatsu Photonics KK, Hamamatsu City, Japan) equipped with scanner software. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining (Roche Diagnostics GmbH, Mannheim, Germany) was performed following the manufacturer's instruction and counterstained with 4′, 6-diamidino-2-phenylindole (DAPI). The alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities of serum samples were measured using a dry chemistry analyzer (FUJI DRI-CHEM 4000ie; Fujifilm Corporation, Tokyo, Japan) according to the manufacturer's instructions.
Nanozoomer 2.0 rs scanner
Manufactured by Hamamatsu Photonics
Sourced in Japan
The NanoZoomer 2.0 RS scanner is a high-resolution digital slide scanning system designed for microscopy applications. It captures detailed images of specimens with up to 40x magnification. The scanner utilizes advanced optics and imaging technologies to provide consistent, high-quality results.
Automatically generated - may contain errors
Lab products found in correlation
Antibody clone d5f3 3633, by Cell Signaling Technology (2 mentions)
Envision flex high ph solution, by Agilent Technologies (2 mentions)
Nanozoomer digital pathology software, by Hamamatsu Photonics (2 mentions)
Dri chem 4000ie, by Fujifilm (1 mentions)
Ndp view software, by Hamamatsu Photonics (1 mentions)
Amh goat primary antibody, by Santa Cruz Biotechnology (1 mentions)
Anti trpv4, by Abcam (1 mentions)
Harris modified hematoxylin, by Thermo Fisher Scientific (1 mentions)
Anti trpv1, by Alomone (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Ultra view hrp system, by Roche (1 mentions)
Anti ki 67, by Roche (1 mentions)
Ndp view2, by Hamamatsu Photonics (1 mentions)
Ventana benchmark ultra ihc ish system, by Roche (1 mentions)
Fluorescently conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Zen digital imaging for lsm 700, by Zeiss (1 mentions)
Zen 2010b sp1 imaging software, by Zeiss (1 mentions)
Anti β tubulin, by Novus Biologicals (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
22 protocols using nanozoomer 2.0 rs scanner
1
Histopathological Analysis of Liver and Ileum
2
Histological Analysis of Lung Tissue
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Lung specimens were fixed in 4% paraformaldehyde, embedded in paraffin, sliced into 5 μm-thick sections, and stained with hematoxylin and eosin according to a standard methodology. Areas of particular concern were analyzed using a NanoZoomer 2.0-RS scanner (Hamamatsu, Shizuoka, Japan).
3
Quantitative Analysis of Immune Cell Markers
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IHC slides were scanned by an Hamamatsu NanoZoomer 2.0 RS scanner (Hamamatsu Photonics, Hamamatsu, Japan) at a magnification of 40× and 20× for morphometric analyses of CD8 and CD56 expression, respectively. Next, morphometric analyses were performed with the use of a Cell^P program (Olympus). To analyze CD8+ cells, the three portal areas were selected for each slide. The surface of the total portal areas and all bile ducts in every portal area were measured. The number of CD8+ cells was counted and calculated per 1 µm2 of both the portal area and the bile duct area. Results were presented as an arithmetical mean ± standard deviation (SD).
To analyze anti-CD56 immunostaining, five regions within the portal area with positive CD56 expression were selected for each slide. The results were presented as a percentage of the area with CD56 expression in selected regions in relation to the entire area of the scanned image under 20× magnification (this area was 147,978.43 µm2). The method of analysis is illustrated inFigure 1 . The threshold parameters were set using the HSI color space model in the following way: hue (H) 90°, saturation (S) 256°, intensity value (I) 123°.
To analyze anti-CD56 immunostaining, five regions within the portal area with positive CD56 expression were selected for each slide. The results were presented as a percentage of the area with CD56 expression in selected regions in relation to the entire area of the scanned image under 20× magnification (this area was 147,978.43 µm2). The method of analysis is illustrated in
4
Lung Injury Assessment Protocol
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Fresh lung tissues were harvested and fixed with 4% paraformaldehyde for more than 24 h, and then embedded in paraffin and sectionalized. Each section was 5 μm thick, and was stained with hematoxylin eosin following the standard protocol. The areas of specific concern were analyzed after sealing by NanoZoomer 2.0-RS scanner (Hamamatsu, Shizuoka, Japan). The lung injury score was measured as previously described [36] (link). Briefly, according to the four independent indicators: pulmonary hemorrhage, neutrophils infiltration, congestion in the pulmonary capillary, and septal thickening, the severity of the lung injury was graded from 0 to 4: 0, normal; 1, mild damage (< 25% injury of the field); 2, moderate damage (25% to 50% injury of the field); 3, severe damage (50% to 75% injury of the field); and 4, extremely severe damage (> 75% injury of the field). The score of each mouse was calculated as the mean of five randomly select fields.
5
Quantification of Ki67 in Lung Arteries
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Randomly chosen lower lobe pulmonary parenchyma portions were fixed in 4 % paraformaldehyde for 48 h, dehydrated and embedded in paraffin. Specific staining for Ki67 (Master Diagnostica, Granada, Spain) and haematoxylin was performed in 4 µm sections. An average size of 200 mm2 for each parenchyma was digitalized using the NanoZoomer 20RS scanner (Hamamatsu Photonics K.K., Hamamatsu, Japan) at a magnification of 40x. The quantification of the positive Ki67 nuclei in the wall of the arteries was performed in the digitalized images using the NanoZoomer digital Pathology viewer (Hamamatsu Photonics K.K., Hamamatsu, Japan). A number of 24 arteries with a diameter between 30 and 100 µm were quantified per subject and the results represented as median (interquartile range) of Ki67 positive nuclei per artery (wall) and also adjusted by the arterial diameter.
6
Quantitative Immunohistochemistry of 14-3-3η in Synovial Tissue
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Formalin-fixed, paraffin-embedded synovial tissue sections (5 µm) were deparaffinized and rehydrated. Immunohistochemical staining was performed according to the standard avidin–biotin immunoperoxidase complex technique [4 (link)], and the sections were incubated with anti-14-3-3η (Augurex, 1:100 in 2% BSA and 2% goat serum) or mouse isotype IgG (DAKO Agilent, Santa Clara, CA, USA,1:100). Diaminobenzidine was used as a substrate for the detection of the labeled proteins, and the sections were counterstained with Harris hematoxylin. Slides were scanned using a Hamamatsu Nanozoomer 2.0-RS scanner. For quantitative immunohistochemistry, six random fields (at original magnification 20X) for each patient were captured using NPD viewing software, and the intensity of labeling in the tissue sections was analyzed using the immunohistochemistry quantification technique as previously described [5 (link)]. The results are expressed as the sum of labeling intensity (density) relative to the total area.
7
Immunohistochemical Analysis of Testicular Cell Types
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Immunohistochemistry was performed on 4% paraformaldehyde-PBS and Bouin solution-fixed, paraffin-embedded tissues, as previously described26 (link). The Sertoli cells were labeled with an AMH goat primary antibody (1:100; Santa Cruz Biotechnology, CA, USA). Leydig cells were stained with a rabbit anti-cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1) antibody (1:250; Sigma Aldrich). A mouse M2A primary antibody (1:100; Abcam, Paris, France) was used for gonocyte immunolabeling. For the AMH and M2A antibodies, antigens were retrieved for 40 min at 80 °C in 10 mM citrate buffer, pH 6.
A NanoZoomer 2.0-RS scanner (Hamamatsu, Tokyo, Japan) was used to capture pictures of the whole slides at 40x magnification. The surface area of 5 to 10 sections randomly selected within the whole explant were calculated with NDP.view software (Hamamatsu). ImageJ software (US National Institutes of Health, Bethesda, MD, USA) was used to perform the stereological cell counting. Germ and Sertoli cells were identified and counted as intra-cordal AMH-negative and AMH-positive cells, respectively.
A NanoZoomer 2.0-RS scanner (Hamamatsu, Tokyo, Japan) was used to capture pictures of the whole slides at 40x magnification. The surface area of 5 to 10 sections randomly selected within the whole explant were calculated with NDP.view software (Hamamatsu). ImageJ software (US National Institutes of Health, Bethesda, MD, USA) was used to perform the stereological cell counting. Germ and Sertoli cells were identified and counted as intra-cordal AMH-negative and AMH-positive cells, respectively.
8
Immunohistochemical Analysis of TRPV Channels in Myometrium
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Paraffin-embedded myometrial tissue sections were freed from paraffin and rehydrated before immunohistochemical staining was performed according to the standard avidin-biotin immunoperoxidase complex technique using the following antibodies: Anti-TRPV1 (Alomone Labs, Jerusalem, Israel; cat#ACC-030; dilution 1:200), anti-TRPV3 (Abcam, Cambridge, UK; cat #ab231150; dilution 1:200), anti-TRPV4 (Abcam; cat #ab39260 dilution 1:200) or rabbit isotype IgG (Dako; Glostrup Denmark; Cat#X0903) as negative control. Diaminobenzidine (DAB; Sigma Fast kit, Sigma-Aldrich; Oakville ON Canada; cat#D4168) was used for the detection of the labeled proteins, and the sections were counterstained with Harris modified hematoxylin (Fisher Scientific, Cat#SH26−500D). Slides were scanned with a Hamamatsu Nanozoomer 2.0-RS scanner from the Histology and Electron Microscopy platform at the Faculty of Medicine and Health Sciences of the Université de Sherbrooke.
9
Immunohistochemical Analysis of Mouse Kidneys
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Mouse kidneys were fixed in 4% paraformaldehyde for at least 24 hours and embedded in paraffin. Paraffin sections were deparaffinized and hydrated in graded ethanol series before staining with the peroxidase-antiperoxidase method. Antigens were retrieved by boiling the sections for 20 min in 10 mM citric acid solution (pH = 6) or 10 mM Tris and 1 mM EDTA (pH = 9). Endogenous peroxidase was blocked by incubation in 3% hydrogen peroxide. The sections were incubated overnight at 4 °C with primary antibodies. The sections were incubated with the corresponding secondary antibodies. 3,3’-diaminobenzidine (DAB) (Sigma-Aldrich, St Louis, MO) was used as chromogen. Sections were lightly counterstained with hematoxylin and were dehydrated and coverslipped. Image acquisition was performed on the Hamamatsu NanoZoomer 2.0 RS scanner.
10
Paraffin-Embedded Mouse Embryo Histology
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Mouse embryos at E16.5 were fixed in 4% PFA overnight, embedded in paraffin, and 5 μm sections were cut and mounted on slides. The sections were deparaffinized by xylene and stained by H&E solution. Nanozoomer 2.0 RS scanner (Hamamatsu) was used to image the slides.
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