According to the manufacturer’s instructions (Boshide, Wuhan, China), the heart and kidney sections were first dewaxed and rehydrated. The endogenous peroxidase activity was blocked with 3% hydrogen peroxidase for 10 min. Then the sections were incubated with goat serum blocking solution at room temperature for 20 min(m). The sections were then incubated with primary antibodies for Apoptosis Stimulating Fragment (FAS, Sigma, St. Louis, MO, USA) or Kidney Injury Molecule 1 (KIM-1, ABCAM, Cambridge, MA, USA), overnight at 4 °C. After being rinsed, the tissue sections were reacted with peroxidase-conjugated goat anti-rabbit IgG secondary antibodies at 37 °C for 30 min. Finally, all sections were dehydrated, cleared, mounted, and visualized with a DAB-based colorimetric method and Image Pro Plus 6.0 analysis software (Media Cybernetics Inc., MD, USA). The IHC index was defined as the average integral optical density.
Image pro plus 6.0 analysis software
Manufactured by Media Cybernetics
Sourced in United States, China
Image-Pro Plus 6.0 is an image analysis software developed by Media Cybernetics. It is designed to assist users in processing, analyzing, and quantifying digital images. The core function of the software is to provide tools for image segmentation, object detection, measurement, and data reporting.
Automatically generated - may contain errors
Lab products found in correlation
Quantity one software, by Bio-Rad (2 mentions)
Kim 1, by Abcam (1 mentions)
3 3 diaminobenzidine tetrahydrochloride, by Vector Laboratories (1 mentions)
Mouse anti rat tgf β1 monoclonal antibody, by Abcam (1 mentions)
Spss software version 19, by IBM (1 mentions)
Chemdoctm xrs, by Bio-Rad (1 mentions)
Horseradish peroxidase conjugated second antibody, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Chemdoc xrs, by Bio-Rad (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Eclipse te2000 s microscope, by Nikon (1 mentions)
Evans blue, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Tak1 antibody, by Merck Group (1 mentions)
Dsx100 optical microscope, by Olympus (1 mentions)
Leica dfc300 fx camera, by Leica camera (1 mentions)
Goat anti rabbit igg fitc, by Proteintech (1 mentions)
Leica dmi4000b, by Leica camera (1 mentions)
23 protocols using image pro plus 6.0 analysis software
1
Immunohistochemical Analysis of Apoptosis and Kidney Injury
2
Immunohistochemical Analysis of α-SMA and TGF-β1
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Specimens were deparaffinized and rehydrated with xylene and 80, 90, 95 and 100% ethanol. Following washing three times with PBS, sections were placed in 0.01 mol/l citrate buffer (pH 6.0), heated in a microwave (92–95°C, 15 min) for antigen retrieval and cooled to room temperature. Following antigen retrieval, primary antibodies, including mouse anti-rat α-smooth muscle actin (α-SMA) monoclonal antibody (Abcam, Cambridge, UK; cat. no. ab7817) and mouse anti rat TGF-β1 monoclonal antibody (Abcam; cat. no. ab64715) were used at dilutions of 1:75 and 1:50, respectively, and incubated overnight at 4°C. Immunoglobulin G-conjugated horseradish peroxidase (HRP) goat anti-mouse polyclonal antibody (1:500; cat. no. ZB-2305; Beijing ZSGB Biotech. Co., Ltd., Beijing, China) and 3,3-diaminobenzidine tetrahydrochloride (Vector Laboratories, Burlingame, CA, USA) were employed to visualize antibody binding (control time under a microscope at room temperature). Positive staining was defined as the presence of tan-yellow color granular staining. Optical density values of positive areas in each slice were calculated using Image-Pro Plus 6.0 analysis software (Media Cybernetics, Inc., Rockville, MD, USA).
3
Protein Expression Analysis Using Image Pro
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For the calculation of protein expression of average optical density (OD), the Image-Proplus 6.0 Analysis Software (Media Cybernetics, Shanghai, China) were used. The obtained data were presented as means ± SD (n = 10). Statistical analysis was performed by the Statistical Program for Social Sciences (SPSS) software version 19.0 (IBM Corporation, Armonk, NY, USA). Analysis of variance (ANOVA) was performed for the multiple comparison of different groups. The histogram was designed by using the software of Graph Pad Prism version 5.0 (San Diego, California, CA, USA).
4
Protein Expression Analysis in Mouse Brain
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Tissue homogenates of the mouse brains and lysates of culture cells were centrifuged at 12,000 rpm for 30 min at 4°C, and quantified for total proteins using the UV 1700 PharmaSpec ultraviolet spectrophotometer (Shimadzu, Japan). Proteins (60 μg) of each sample were separated on 10% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, CA, USA) using an electronasfer device (45 V, 15 h). After blocking in 5% fat-free milk in TBS containing 0.1% Tween-20 for 1 h, the membranes were then incubated with the following primary antibodies: anti-DMT1-IRE (1:2000, Alpha Diagnostic, San Antonio, TX, USA), anti-DMT1-nonIRE (1:1000, Alpha Diagnostic, San Antonio, TX, USA), anti-Ndfip1 (1:2000), anti-APP695 (1:4000, Chemicon, USA), and anti-GAPDH (1:10,000, KC-5G5, Kang Chen, China) for 2 h at room temperature. Then the membranes were washed and incubated with horseradish peroxidase-conjugated second antibody (1:5000, Santa Cruz, CA, USA) for 2 h at room temperature. Immunoreactive bands were visualized by an enhanced chemiluminescence kit (Pierce, Appleton, WI, USA) and ChemDocTM XRS with Quantity One™ software (BioRad, USA). Blots were repeated at least three times for every condition. The band intensities were quantified by Image-pro Plus 6.0 analysis software (Media Cybernetics, Rockville, MD, USA).
5
Ghrelin and Cell Proliferation Analysis
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Ghrelin and PCNA labeling were examined by a microscope (Olympus BX41 (DP25), Japan). From each specimen, views of the fields in 10 stained sections were randomly chosen (n = 10 sections, per treatment) and photographed using the Olympus microscope camera system (Olympus BX41 (DP25), Japan). The next step was using Image Pro-Plus 6.0 analysis software (Media Cybernetics, MD, Maryland, USA) to analyze and evaluate the amount of immuno-positive cells stained. The SIOD (Integrated Optical Density of the single small intestinal villi) and IOD (Total Integrated Optical Density of a cross-section) were obtained, which were applied to compare the ghrelin and PCNA staining intensities in the duodenum, jejunum and ileum between the control and ZEA treatment.
6
Western Blotting of Brain Tissue Proteins
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Brain tissue samples were homogenized in a buffer containing 40 mM Tris-HCl (pH 7.0), 1% Triton X-100, 0.2% SDS, 1.0 mM sodium deoxycholate, 1.0 mM EGTA, 1.0 mM EDTA, 1.0 mM Na3VO4, 50 mM NaF, 1.0 mM PMSF, and 2.0 μg/ml each of aprotinin, leupeptin and pepstatin. The homogenates were centrifuged at 16,000 rpm for 15 min, and the supernatants were stored in aliquots at -80°C until use. The tissue extracts were separated in SDS-polyacrylamide gels and then transferred onto polyvinylidene difluoride membrane (Millipore, CA). Nonspecific binding sites on the membrane were blocked by 5% bovine serum albumin in 0.1% TBS/Tween-20 (TBST) for 1 h. The membrane was then incubated with specific primary antibodies (Table 1 ) overnight at 4°C. After washing with TBST, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (1:2000; Cell signaling Technology, Beverly, MA) for 2 hours at room temperature. Immunoreactive bands were visualized by using the Super Signal West Pico Chemi-luminescent Substrate (Pierce Biotechnology, Rockford, IL) and Chem Doc XRS with Quantity One software (BioRad, USA). The protein bands were scanned and the intensities of the bands were measured using Image-pro Plus 6.0 analysis software (Media Cybernetics, Rockville, MD, USA).
7
Histological Analysis of Renal Tissue
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For histological analysis, fresh left renal tissue collected from each group was fixed using 4% paraformaldehyde for 12 h at 4°C. Tissues were embedded in paraffin, cut into 5-µm thick section and stained using hematoxylin for 5 min and 0.5% eosin for 2 min (H&E) at room temperature. For Masson's trichrome staining, paraffin sections were dewaxed with xylene, rehydrated with graded ethanol at room temperature and stained with Regaud dye hematoxylin for 10 min at room temperature. Following rinsing with water, sections were stained with Ponceau Fuchsin acid solution for 10 min at room temperature, immersed in 2% acetic acid aqueous solution and treated with a 1% aqueous solution of phosphomolybdic acid for 5 min at room temperature. Without rinsing with water, the sections were stained with 2% aniline blue for 5 min at room temperature, immersed in 0.2% acetic acid aqueous solution, in 95% alcohol, anhydrous alcohol, permeabilized with xylene and mounted with neutral resin. The sections were observed and images were captured using a NanoZoomer 2.0 RS Digital Pathology slide scanner (Hamamatsu Photonics K.K., Hamamatsu, Japan). Renal collagen volume fraction (CVF) was analyzed using Image-Pro Plus 6.0 analysis software (Media Cybernetics, Inc., Rockville, MD, USA). A total of five fields of view per sample were randomly chosen and the average was determined for analysis.
8
Quantifying MMP-1 Expression in Lung Tissue
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Tumor-adjacent lung tissues were obtained from 28 surgery patients in our cohort prior to radiotherapy, and then fixed in 10% formalin overnight, paraffin-embedded, and sectioned at an average thickness of 4 μm. Deparaffinized sections were subjected to antigen retrieval and then incubated with 3% hydrogen peroxide for 15 min. After blocking with 5% bovine serum albumin, the sections were incubated with anti-MMP-1 antibody (AP11874c,1:50, Abgent, San Diego, CA, USA) overnight at 4°C, followed by incubation with secondary goat anti-rabbit antibody (GB23303; Wuhan Goodbio Technology Co., Wuhan, China) for 50 min. Counterstaining with hematoxylin was performed, and 3,3′-diaminobenzidine positivity was analyzed. Images were captured using an Eclipse TE2000-S microscope (Nikon, Tokyo, Japan). The mean optical density obtained from alveolar areas for each stained slide was quantified by Image-Pro Plus 6.0 analysis software (Media Cybernetics, Rockville, MD, USA).
9
NASH Liver Tissue Characterization
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A total of 18 NASH patients' (10 males and 8 females) liver tissues were collected from the Eastern Hepatobiliary Surgery Hospital (Shanghai, China) according to the guidelines between 2010 and 2019 13 (link). Blood routine parameters, including white blood cells, red blood cells, and biochemical markers [total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST)] were showed in the table (Supplemental Table 1 ). Four control liver tissue sections were collected from normal tissue adjacent to hepatocellular carcinoma by liver cancer resection. Written informed content was obtained from all subjects prior to participation in the study. The tissue samples of NASH were diagnosed by a pathologist and collected according to an established protocol approved by the Ethics Committee of Eastern Hepatobiliary Surgery Hospital. The immunohistochemistry (IHC) staining of AGK in liver sections used rabbit-anti human AGK antibody (Invitrogen, Carlsbad, CA, USA). The staining intensity was analyzed using Image Pro Plus 6.0 analysis software (Media Cybernetics, Rockville, MD, USA).
10
Measuring Myocardial Infarct Size
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After reperfusion in each group, the animals were euthanized by cervical dislocation. The rat hearts were removed, and 1 ml 3% Evans blue (Sigma-Aldrich; Merck KGaA) was injected into the left ventricle. Perpendicular to the long axis direction of the heart, the heart tissue was cut into thin slices with a thickness of 5 µm and 1% 2,3,5-triphenyltetrazolium chloride solution was incubated with the heart tissue section at 37˚C for 15 min. Image Pro Plus 6.0 analysis software (Media Cybernetics, Inc.) was used to determine the infarct size.
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