F-actin bundles thickness were measured with Image-Pro-Plus. Merge of LMO7 and cortical F-actin in NRK-52E cells that underwent hypertonic-isotonic alternation for two hours was calculated the yellow color area as numerator and cortical F-actin as denominator. Ratio of LMO-7/F-actin merge to F-actin was calculated. All images were analyzed with software Image Pro Plus 8.0 (Media Cybernetics, Rockville, MD, USA).
Image pro plus 8
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Sourced in United States
Image-Pro Plus 8.0 is a comprehensive image analysis software that provides advanced tools for image capture, processing, measurement, and analysis. It offers a wide range of features to support scientific and research applications.
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Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Incucyte live cell analysis system, by Sartorius (2 mentions)
Sp rabbit mouse hrp dab kit, by CWBIO (1 mentions)
Inverted microscope, by Media Cybernetics (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Abcam (1 mentions)
Eclipse ti2, by Nikon (1 mentions)
10 protocols using image pro plus 8
1
Measuring F-actin Bundles and LMO7/F-actin Ratio
2
Comprehensive Liver Fibrosis Evaluation
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Masson’s trichrome staining, Sirius red, H&E, immunohistochemical (IHC), and IF staining, and double IF staining were performed as previously described [3 (link)]. Masson’s trichrome staining and Sirius red staining were used for collagen investigation, and semiquantitative analysis was performed using Image-Pro Plus 8.0 (Media Cybernetics, Rockville, MD, USA). For IHC, after antigen retrieval, 3 μm-thick paraffin-embedded sections were permeabilized with 1% Triton X-100, followed by PBS washes and incubation with the indicated antibodies overnight at 4 °C. Next, the targeted protein was detected using a secondary antibody and then incubated in DAB for intensification, and the sections were counterstained with hematoxylin. For IF staining, the targeted proteins were incubated with the indicated primary antibodies overnight at 4 °C, followed by the related biotin-conjugated secondary antibody and streptavidin Alexa Fluor 488® or 594® (Thermo Fisher Scientific). The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). For double staining, after completing the first protein staining, the sections were then re-incubated, and the secondary protein was detected. The liver fibrosis stage was assessed by the Ishak scale [60 (link)].
3
Quantifying Aortic Endothelial CD31 Expression
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At the age of 19 weeks, ApoE−/− and ApoE−/−CD137−/− mice were euthanized by cervical dislocation, and thoracic aorta was removed. Serial sections on the same paraffin blocks were applied to perform immunohistochemistry staining by SP Rabbit & Mouse HRP Kit (DAB) (CWbiotech). The slides were boiled for 10 minutes with EDTA (ethylene diamine tetraacetic acid) antigen retrieval buffer, blocked with normal goat serum, and incubated with primary antibody for CD31 (1 : 100, Abcam) at 4°C overnight. The following day, the slides were treated with biotin-linked goat anti-rabbit/mouse IgG, followed by Streptavidin-HRP. DAB chromogen solution was used to develop the color (positive cells stained brown), while hematoxylin was applied to counterstain cell nuclei. Brown areas were calculated using Image-Pro Plus 8.0 (Media Cybernetics).
4
Clonogenic Assay for M1 Radiosensitivity
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The HIEC-6 cells were planted in a 6-well plate with a density of 500 cells/well for 24 h. After attachment, the cells were incubated with the solution of M1 at different concentrations of 0.1 and 0.5 mM prior to irradiation (0, 4, 6, 8, and 10 Gy). Ten days after irradiation, the cells were fixed with 1 mL paraformaldehyde (4%) for 60 min and stained with crystal violet (0.5%) for 10 min, and then, the cells were washed with distilled water twice after discarding the dye solution. The number of colonies was calculated by Image Pro Plus 8.0 software (Media Cybernetics, Maryland, USA).
5
Wound Healing Assay with SSA Treatment
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HUVECs were seeded in 96-well plates (1 × 104 cells/well) and allowed to grow to confluence. Then the cells were scratched with WoundMaker (IncuCyte) and treated with various concentrations of SSA (1–100 μM) for 12 h. Then the cells were incubated with fresh medium till 48 h. After 48 h, the cells were observed and photographed using the IncuCyte Live-Cell Analysis System (Essen BioScience). Cell migration was quantified using Image-Pro Plus 8.0 software (Media Cybernetics, Bethesda, MD).
6
Wound Healing Assay with JuB
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HUVECs were seeded in a 96-well plate (three replicates per group) at a density of 1 × 104 cells/well. Then, the Wound Maker (IncuCyte) scratched the HUVECs. The cells were then washed with PBS and cultured with various concentrations of JuB (1–100 μM) for 12 h. Then the cells were incubated in fresh medium till 48 h, and the cells were photographed using the IncuCyte Live-Cell Analysis System (Essen BioScience). The closure area of the wound was quantified using Image-Pro Plus 8.0 software (Media Cybernetics, Bethesda, MD).
7
Western Blot Analysis of CHD1L in BGC-823 Cells
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Lentivirus-infected BGC-823 cells were harvested and rinsed three times, and total protein was extracted using RIPA-buffer. Protein concentration was measured using the BCA protein concentration detection kit. Proteins (60 µg) were isolated by 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked in 5% skim milk powder-TBST for 2 h at room temperature and incubated with primary anti-CHD1L antibody (1:1,000; cat. no. sc81065) and monoclonal β-actin antibody (1:1,1000; cat. no. sc58673; Santa Cruz Biotechnology, Inc.) at 4 °C overnight. Membranes were rinsed with TBST three times and incubated with secondary antibody [1:2,000; HRP-conjugated-goat anti-rabbit polyclonal IgG; cat. no. D110058, Sangon Biotech (Shanghai) Co., Ltd., China] at 37 °C for 2 h. An ECL chemiluminescence kit was used to visualize protein bands. Images were analyzed with Image-Pro Plus 8.0 software (Media Cybernetics, Inc.), and the ratio of CHD1L to β-actin was determined. The CHD1L-shRNA with the highest knockdown efficiency was used in subsequent experiments.
8
Cell Migration Quantification Assay
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Cell migration was evaluated using a scratch (wound) healing assay as in our previous study [20 (link)]. In brief, HUVEC monolayers were wounded with a 200 μL pipette tip. Cells were then maintained for 8 h in basal medium containing PBS or Ang-1 (300 ng/mL). Wounded areas were visualized with an Olympus inverted microscope and quantified using Image-Pro Plus™ 8.0 software (Media Cybernetics, Bethesda, MD, USA). Values are reported as percent wound healing, which were calculated using the following formula:
where t8 is the time (8 h) over which cells were maintained in the medium and t0 is the time immediately after wounding.
where t8 is the time (8 h) over which cells were maintained in the medium and t0 is the time immediately after wounding.
9
Histological Analysis of EAT
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EAT was embedded in paraffin and cut into 4 μm sections for hematoxylin and eosin (H&E) staining. Sections were also treated with mouse anti-human leptin monoclonal antibody (Novus Biologicals, USA), rabbit anti-human CTRP9 antibody (Aviscera Bioscience, USA), or mouse anti-human macrophage antibody (Santa Cruz, USA). After visualization, these sections were observed under a microscope and representative images were analyzed with Image-Pro Plus 8.0 software (Media Cybernetics, Cambridge, MA, USA).
10
Investigating DNA Damage Using γ-H2AX Assay
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Histone H2AX (γ-H2AX) was used to investigate DNA double strand breaks (DSBs). The HIEC-6 cells were incubated with a density of 10 × 105 cells per confocal microscope dish. After attachment, the cells were co-incubated with M1 for 30 min before irradiation. Then, 1 h after irradiation, the cells were fixed with 4% paraformaldehyde for 20 min and then were washed with PBS and treated with 0.2% Triton-X 100 for 15 min. After PBS washing, the cells incubated with rabbit polyclonal γ-H2AX (phospho S139) primary antibody (dilution 1: 1000; cat. No. ab2893; Abcam, Cambridge, MA, USA) at 4 °C overnight. After discarding the primary antibody, the cells were gently washed with PBS three times and were incubated with goat anti-rabbit secondary antibody (diluted 1:2000; cat. No. ab6939; Abcam) at room temperature for 1 h. The nuclei were counterstained with DAPI (cat. No. C0065, Solarbio, Beijing, China). Images of the cells were collected using a confocal microscope under a 63 × oil microscope (Nikon, Eclipse Ti2, Tokyo, Japan). The foci in each picture were analyzed by Image Pro Plus 8.0 software (Media Cybernetics, Maryland, USA).
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