Paraffin blocks were sectioned (5 μm) and placed on poly-L-lysine-coated glass slides. The sections were deparaffinized for 1 h at 60°C. To block the activity of endogenous peroxidase, the sections were incubated in methanol containing 0.3% H2O2 for 30 min. Thereafter, every step was followed by three washes in PBS buffer. To block nonspecific antibody binding, slides were incubated for 1 h in 1% BSA in PBS. Sections were incubated with galectin-9 antibody (ab69630, Abcam, Cambridge, MA USA) diluted at 1:400 overnight at 4°C. Then, the sections were incubated with HRP-conjugated anti-rabbit IgG (ab6721, Abcam) for 1 h at room temperature. Staining was visualised by incubating the slides with DAB substrate (ImmPACT DAB Peroxidase substrate, Vector SK-4105, Burlingame, CA USA) according to the manufacturer’s instructions. To distinguish the nuclei, sections were counterstained in haematoxylin solution (HX87960674 Merck, Darmstadt Germany) for 10 sec. Images were obtained using a VENTANA DP200 scanner (Roche Diagnostics, Tucson, AZ USA). The intensity of galectin-9 expression was determined using Image-Pro Image Analysis Software (Media Cybernetics, Rockville, MD USA). The mean density was calculated for each sample and the expression of galectin-9 was reported in lumens.
Imagepro image analysis software
Manufactured by Media Cybernetics
Sourced in United States
ImagePro is an image analysis software developed by Media Cybernetics. It provides tools for processing, analyzing, and interpreting digital images.
Automatically generated - may contain errors
Lab products found in correlation
Dcfh da, by Beyotime (2 mentions)
Ab69630, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Immpact dab peroxidase substrate, by Vector Laboratories (1 mentions)
Ventana dp200 scanner, by Roche (1 mentions)
Ultrafast scanner, by Philips (1 mentions)
Fluorescence detection kit, by Roche (1 mentions)
Dp80 microscope, by Olympus (1 mentions)
Maguafire sp digital camera, by Olympus (1 mentions)
Fv1000, by Olympus (1 mentions)
Mitosox fluorescence probe, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Inspeck microspheres, by Thermo Fisher Scientific (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Eosin solution, by Merck Group (1 mentions)
Mitosox, by Thermo Fisher Scientific (1 mentions)
M20036, by Thermo Fisher Scientific (1 mentions)
Laser confocal petri dishes, by Corning (1 mentions)
Fluoview fv1000 microscope, by Olympus (1 mentions)
12 protocols using imagepro image analysis software
1
Galectin-9 Immunohistochemistry Protocol
2
Quantification of Lung Metastatic Burden
3
TUNEL Assay for Myonuclei Apoptosis
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TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assessment of myonuclei positive for DNA strand breaks was determined using a fluorescence detection kit (Roche Applied Science, Indianapolis, IN) and fluorescence microscopy. After dewax and rehydrate, paraffin tissue sections were permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 8 min on ice. TUNEL reaction mixture containing terminal deoxynucleotidyl transferase (TdT), fluorescein-dUTP was added to the sections in 50-µl drops and incubated for 60 min at 37 °C in a humidified chamber in the dark. The sections were rinsed three times in PBS for 5 min each. Following embedding, sections were visualized with an Olympus DP80 microscope equipped with an Olympus MaguaFire SP digital camera. DNase I and label solution were used as positive and negative controls. To determine the percentage of apoptotic cells, the TUNEL-positive nuclei and TUNEL-negative cells were counted using the Image-Pro image analysis software (Media Cybernetics, Bethesda, MD)62 (link).
4
Mitochondrial Membrane Potential Measurement
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Mitochondrial membrane potential was measured using the fluorescence probe TMRM (Invitrogen) according to the manufacturer’s protocol. Cells were incubated with 10 nM TMRM for 10 min at 37 °C in the dark and images were captured by laser confocal microscope (FV1000, Olympus) and analyzed by ImagePro image analysis software (Media Cybernetics, Silver Spring, MD, USA).
5
Measuring Cellular and Mitochondrial Reactive Species
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Cellular ROS and mitochondrial reactive species (mitoROS) were detected using the fluorescence probe DCFH-DA (Beyotime Institute of Biotechnology, China) and mitoSOX fluorescence probe (Invitrogen; Thermo Fisher Scientific, Inc., USA), respectively, following the protocols described previously.10 (link) ImagePro image analysis software (Media Cybernetics, Inc., USA) was used to capture and process the images.
6
Quantifying Apoptosis in Mouse Cardiac Tissue
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Left ventricular tissue was isolated from mice and fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 5-μm serial sections. Sections were stained using the TUNEL apoptosis kit (Genmedscientfics, MA, USA) according to standard procedures. Briefly, sections were incubated with TUNEL reaction mixtures for 1 h at 37°C followed by two PBS washes (5 min/time). Then, sections were blocked with glycerol buffer and photographed under a fluorescence microscope (Olympus, Tokyo, Japan). To determine the percentage of apoptotic cells, TUNEL-positive and TUNEL-negative cells were counted using the ImagePro image analysis software (Media Cybernetics, MD, USA). Five fields (500 × 500 μm for each field) per section were randomly selected at ×400 magnification. The analyses were performed in a blinded manner.
7
Measurement of Intracellular Superoxide in Cardiomyocytes
8
Histopathological Analysis of Articular Cartilage
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After 4 weeks of continuous feeding, mice were intraperitoneally anaesthetized with 1% sodium pentobarbital and immediately sacrificed. Then, the knee joints were fixed in 4% paraformaldehyde for 24 h, and treated with ethylenediaminetetraacetic acid (EDTA) to decalcification for seven days. The joints were embedded in paraffin, and 7 mm sections were taken for haematoxylin–eosin (H&E) staining (Kim et al. 2020 ). Specifically, the paraffin sections were deparaffinized, alcohol hydration was graded, haematoxylin solution was stained for 20 min, and eosin solution was counterstained (Sigma-Aldrich, St. Louis, MO) for 10 min. After gradient ethanol dehydration, transparent and sealed, the image-Pro image analysis software (Media Cybernetics, Rockville, MD) was used to observe the pathological changes of articular cartilage.
9
Quantifying Virus-Induced Syncytia Formation
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Monolayers of DBT-WT, DBT-ERp29, and DBT-EGFP cells grown on coverslips were infected with MHV-A59 at MOI 2 for 6 h, 9 h, and 12 h. Also, DBT-WT cells were treated with 4-PBA p.i. with MHV-A59. Cells were immunostained for viral N and subsequently fixed with 4% PFA and mounted on glass slide using Vectashield with DAPI to stain all nuclei. The cells were then visualized using an Olympus IX-81. Images were acquired using an Orca-1 charge-coupled device and camera (Hamamatsu) and Image-Pro image analysis software (Media Cybernetics, Silver 99 Spring). Syncytia formation was quantified for each condition, from three independent biological sets, in terms of fusion index using the following formula: Fusion Index = (N-S)/T × 100, where N denotes the number of nuclei in the syncytia, S indicates the number of syncytia, and T denotes the total number of nuclei (58 (link), 59 (link)). Kinetics of virus-induced cell–cell fusion was determined by plotting the fusion index values against the respective times in GraphPad Prism.
10
Quantifying Cellular and Mitochondrial ROS
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Fluorescence probe DCFH-DA (Beyotime, Beijing, China) was used to detect cellular reactive oxygen species (ROS). The fluorescence intensity was assessed by flow cytometry with an excitation wavelength of 488 nm and an emission wavelength of 535 nm. The fluorescence probe mitoSOX (Invitrogen) was used to detect mitochondrial reactive oxygen species (mitoROS) under microscopy (FluoView 1000). Images were captured and analyzed by ImagePro image analysis software (Media Cybernetics, Silver Spring, MD, USA).
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