Tumor samples were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned into 4-μm-thickness slices. After dewaxing, tissues sections were processed by antigen retrieval, followed by the quenching of endogenous peroxidase activity using hydrogen peroxide. PBS (0.1% Tween 20) containing normal serum was used to block non-specific binding sites. Slides were incubated with rabbit polyclonal antibodies against CD31 or VEGF-A (in 1:50 dilution, Santa Cruz, US). After washing with PBS, slides were incubated with biotinylated anti-rabbit IgG antibody followed by horseradish peroxidase-conjugated streptavidin (Sigma, US). After developing in DAB substrates (Invitrogen, US), 5 high-magnification fields (400×) were randomly selected in each slide, and the numbers of positive cells in each field were counted by the image analysis system (Image-Pro Plus). In a parallel negative control group, PBS was applied to replace the primary antibody.
Image pro plus
Manufactured by Thermo Fisher Scientific
Sourced in United States
Image-Pro Plus is a powerful image analysis software developed by Thermo Fisher Scientific. It provides a comprehensive suite of tools for capturing, processing, analyzing, and managing digital images across a variety of research and industrial applications.
Automatically generated - may contain errors
3 protocols using image pro plus
1
Immunohistochemical Quantification of Angiogenic Markers
2
Immunohistochemical analysis of tumor markers
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Tumor samples were fixed in 4% formalin, embedded in paraffin, and sectioned into 4-μm-thickness slices. After dewaxing, tissues sections were processed by antigen retrieval, followed by the quenching of endogenous peroxidase activity using hydrogen peroxide. PBS (0.1% Tween 20) containing anti-goat serum was used to block non-specific binding sites. Slides were incubated with polyclonal antibodies against Ki-67 or VEGF-A or MMP-9 (in 1:100 dilution, CST, US). After washing with PBS, slides were incubated with biotinylated anti-rabbit IgG antibody followed by horseradish peroxidase-conjugated streptavidin. After developing in DAB substrates (Invitrogen, US), 5 high-magnification fields were randomly selected in each slide, and the numbers of positive cells in each field were counted (each group=4) by the image analysis system (Image-Pro Plus).
3
Histopathological Evaluation of Cardiac Tissue
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Hearts were fixed in 10% formalin and embedded in paraffin using standard histological procedures. Subsequently, these hearts were sectioned transversely at 5 μm. Sections were stained with hematoxylin-eosin for histopathology or with PSR to evaluate collagen deposition. The myocyte cross sectional area was measured using a quantitative digital image analysis system (Image-Pro Plus, version 6.0) from images captured from FITC-conjugated wheat germ agglutinin (WGA, Invitrogen) stained sections. More than 100 myocytes in the sections were outlined in each group.
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