For the liver and kidneys, sections from each level were stained with hematoxylin (Sigma, H-3136-25G) and eosin (Sigma, E4009-5G) (H&E), Masson (Beijing Solarbio Science & Technology Co., Ltd. G1340), and PAS (Beijing Solarbio Science & Technology Co., Ltd. G1280). The images were acquired using an Olympus VS120 microscope and × 20 objective with 0.5 NA and × 40 objective with 0.75 NA (Olympus VS120-SL). One section from each tissue sample was selected, and three random fields of per slice were quantified. The areas of inflammation and fibrosis in the liver and kidney were measured using the ImageJ software, and the average area of three fields in the same slice was used for statistical analysis.
Vs120 sl
Manufactured by Olympus
The VS120-SL is a slide-loading virtual slide scanning system designed for high-throughput digitization of histological samples. It captures high-resolution images of entire glass slides and enables efficient digital archiving and analysis of microscopic specimens.
Automatically generated - may contain errors
Lab products found in correlation
Ab16669, by Abcam (1 mentions)
Ab64100, by Abcam (1 mentions)
Ab3523, by Abcam (1 mentions)
Ab6640, by Abcam (1 mentions)
Ab5320, by R&D Systems (1 mentions)
F ab 2 fragment alexa fluor 555 conjugate, by Cell Signaling Technology (1 mentions)
Vs120 microscope, by Olympus (1 mentions)
F ab 2 fragment alexa fluor 488 conjugate, by Cell Signaling Technology (1 mentions)
Ammonia water, by Sinopharm (1 mentions)
Sodium iodate, by Merck Group (1 mentions)
Mouse tnf α elisa kit, by Abcam (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Mouse il 6 elisa kit, by Abcam (1 mentions)
Orange g, by Merck Group (1 mentions)
Alcian blue, by Merck Group (1 mentions)
Tartaric acid, by Merck Group (1 mentions)
Mvx10, by Olympus (1 mentions)
Hydrochloric acid (hcl), by Sinopharm (1 mentions)
Xylene, by Sinopharm (1 mentions)
Ginsenoside rg1, by Chengdu Pufei De Biotech (1 mentions)
7 protocols using vs120 sl
1
Histological Analysis of Liver and Kidney
2
Immunostaining of Kidney Tissue Sections
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The sections were blocked by 5% BSA and then incubated overnight at 4 °C with primary antibodies including anti-NG2 chondroitin sulfate proteoglycan (Millipore, AB5320), mouse nephrin antibody (R&D Systems, AF3159), anti-CD45R (Abcam, ab64100), anti-CD3 (Abcam, ab16669), anti-iNOS (Abcam, ab3523), and anti-F4/80 (Abcam, ab6640). After washing with phosphate buffer, the sections were incubated for 1 h at room temperature with the secondary antibodies as follows: anti-rabbit IgG (H + L), F(ab’)2 Fragment (Alexa Fluor® 488 Conjugate) (Cell signaling, #4412), anti-rat IgG (H + L) (Alexa Fluor® 488 Conjugate) (Cell signaling, #4416), anti-rabbit IgG (H + L) F(ab’)2 Fragment (Alexa Fluor® 555 Conjugate) (Cell signaling, #4413). Images were acquired using an Olympus VS120 microscope and × 20 objective with 0.5 NA and × 40 objective with 0.75 NA (Olympus VS120-SL). At least five animals were analyzed in each group, one section of each mouse was selected, and three random fields of each section were analyzed. The data were expressed as the average number of positive cells per field.
3
Automated Quantification of Bone Marrow
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Whole-slide images were acquired with an automated slide scanner (Olympus VS120-SL) at 20x magnification with focus points along the total area of interest or using the in-situ focus mode using the accompanying software (Olympus VS-ASW L100 2.9). VSI files obtained from scanning were directly loaded into QuPath for analysis on Windows or Mac operating systems. Extracted TIFF files were also used for analysis. The “training-set” consisted of 79 images of full or partial bones. The “validation-set” consisted of 59 images of full bones. Parameters are pre-set but adjustable by the user if deemed necessary (e.g., adipocyte circularity or size, see tutorial for details). The software workflow follows basic morphological operations, color deconvolutions, thresholding, smoothing operations, and watershed as annotated in the code provided as open source and described in the technical guidelines. When accounting for pre-processing steps such as the drawing of the regions of interests as well the as the segmentation process, it typically takes 1–2 min in order to obtain MarrowQuant outputs for an image with a sample of a mouse bone. Technical details are annotated in the code and details on download, installation and use of the plugin with the QuPath software are given in the tutorial (MarrowQuant Tutorial, Supplementary Material ).
4
Ginsenoside Rg1 Effects on Joint Histology
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Following 12 weeks of treatment with ginsenoside Rg1, the knee and ankle joints of the mice were harvested, and the specimens were fixed in 10% formalin for 24 h. Then the specimens were continuously washed in water for 2 h and transferred to 10% ethylene diamine tetraacetic acid (EDTA) decalcification solution for 20 days. The decalcification solution was replaced once every 48 h. After decalcification, the specimens were washed in water continuously for 2 h, dehydrated with a series of ethanol, and embedded in paraffin. After paraffin embedding, serial sagittal sections of 4 μm were obtained and stained with HE, alcian blue/orange G (ABOG), and tartrate-resistant acid phosphatase (TRAP). After a full-length scan using an Olympus VS120-SL, the target area of the ankle talus and knee joints were analyzed with Olympus VS120 image analysis software. The cartilage (blue zone) and synovial inflammation areas were measured in the ABOG-stained sections, and TRAP-positive areas were evaluated in the TRAP-stained sections.
5
Histological Analysis of Mouse Ankle Joints
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After final treatment, the mice were sacrificed. Ankle joints were carefully dissected and cleared of adjacent muscle, followed by being fixed in 4% paraformaldehyde at room temperature for 48 h, decalcified in 10% EDTA for 20 d, dehydrated through a series of ethanol, and embedded in paraffin. A total of 30 4-μm-thick consecutive sections from one ankle joint in the sagittal position were collected and mounted on common slides, and were divided into three levels. Each level was 40 μm from the previous level. One section from each of the three levels was subjected to hematoxylin and eosin (H&E) staining, Alcian Blue/Orange G (ABOG) staining, tartrate-resistant acid phosphatase (TRAP) staining, and immunofluorescence and immunohistochemical staining. After the full-length scan by Olympus VS120-SL, the target area of the ankle talus was analyzed with Olympus VS120 image analysis software. We traced the area of interest, which was then measured by the software (in mm2). The inflammatory area (multinuclear dark zone) and bone area (astragalus bone zone) were analyzed on HE-stained sections. Cartilage area (blue zone) at astragalus bone was measured on ABOG-stained sections, and TRAP-positive area was evaluated on TRAP-stained sections. The data are presented as the mean from three levels from each ankle joint sample.
6
Ginsenoside Rg1 in Murine Inflammation
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Main reagents: Ginsenoside Rg1 (Chengdu Pufei De Biotech Co. Ltd., 22427-39-0), Isoflurane (Reward, 217140901), indocyanine green (Akorn, 17478-701-02), 10% formalin (Guangzhou Vigers Biotechnology, 14071005), xylene (Sinopharm Chemical Reagent Co. Ltd., 20140506), hydrochloric acid (Sinopharm Chemical Reagent Co. Ltd., 20140228), ammonia water (Sinopharm Chemical Reagent Co. Ltd., 20140607), phosphate buffered saline (PBS) (Medicago, 09-2052-100), hematoxylin (Sigma, H-3136), eosin (Sigma, E4009-5G), Alcian Blue (Sigma, A5268), orange G (Sigma, 1936-15-8), AS-BI phosphate (Sigma, 1919-91-1), ammonium aluminum sulfate (Sigma, A-2140), sodium iodate (Sigma, S-4007), tartaric acid (Sigma, 87-69-4), Mouse IL-6 ELISA Kit (Abcam, ab100713), Mouse TNF-α ELISA Kit (Abcam, ab208348).
Main instruments: Spray anesthesia machine (Matrx, 07149), NIR imaging system (Olympus, MVX10), and Olympus fully automated tissue scanner (Olympus, VS120-SL).
Main instruments: Spray anesthesia machine (Matrx, 07149), NIR imaging system (Olympus, MVX10), and Olympus fully automated tissue scanner (Olympus, VS120-SL).
7
Decalcification and Histological Analysis
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Use 14% ethylenediaminetetraacetic acid (pH 7.4) was applied for decalcification for 3–4 weeks. After confirming the decalcification by X‐ray, dehydration and paraffin embedding were performed, and sagittal serial sections (4 μm) were performed. Decalcified femoral sections were stained with haematoxylin and eosin (HE) and Alcian Blue/Haematoxylin and Orange G (ABHO), collected, and analysed using Olympus VS120‐SL.
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