Image pro plus software version 4

Manufactured by Media Cybernetics

Sourced in United States

Image Pro Plus software version 4.5 is a comprehensive image analysis software for various scientific and medical applications. It provides a suite of tools for capturing, processing, measuring, and analyzing digital images. The software is designed to facilitate quantitative analysis of images, enabling users to extract valuable data and insights from visual information.

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Lab products found in correlation

Hydroxyapatite coated plate, by Corning (3 mentions) Phalloidin, by Thermo Fisher Scientific (2 mentions) Bovine serum albumin (bsa), by Merck Group (2 mentions) Leica rm2145 microtome, by Leica Microsystems (2 mentions) Fv1000, by Olympus (2 mentions) Sp 9002 histostain plus kits, by ZSGB-BIO (2 mentions) Smz 168, by Motic (2 mentions) Binocular light microscope, by Zeiss (1 mentions) Bx50 microscope, by Olympus (1 mentions) Sodium hydroxide (naoh), by Merck Group (1 mentions) Triphenyltetrazolium chloride, by Merck Group (1 mentions) Leica microscope, by Leica Microsystems (1 mentions) Axio scan z1 scanner, by Zeiss (1 mentions) Zen blue edition, by Zeiss (1 mentions) Ab37647, by Abcam (1 mentions) Ab58968, by Abcam (1 mentions) Ab6671, by Abcam (1 mentions) Ab9324, by Abcam (1 mentions) Coolsnap pro, by Media Cybernetics (1 mentions) Ds qi2, by Nikon (1 mentions)

30 protocols using image pro plus software version 4

Histological Analysis of Organ Tissues

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The tissues were immersed in 10% formaldehyde solution for 24 hours. After the fixation period, they were subjected to dehydration and inclusion in paraffin blocks, which were then sliced to 4-μm thick sections. Histological sections of the heart, lung, kidney, and liver were obtained and the parameters studied were rated from 0 to 3 (0: absence of findings; 1: discreet findings; 2: moderate findings; 3: intense findings) (Table 1). The sums of all scores for each organ were used for comparison.
The analyses were performed by optical microscopy with the aid of the Image Analyzer system. The workstation consisted of a Zeiss Binocular Light microscope, an image digitizing board, and a microcomputer with a Pentium processor, Windows^©. The images were obtained with the help of Image Pro Plus software version 4.5 (Media Cybernetics^©), specific for image analysis so that the different variables were quantified and the results transferred to a spreadsheet (Microsoft Excel^©). The histological sections, in turn, were analyzed using 10 photos of each slide acquired at a magnification of 200x (20x objective and 10x eyepiece). All measurements were archived in a computer-specific program (SPSS-15.0, SPSS inc^©) for subsequent statistical analysis.

de Moraes P.A., Tannuri A.C., Rios L.M., Paes V.R., Gonçalves J.D., Serafini S, & Tannuri U. (2020). Sepsis and cirrhosis in growing animals: description of a new experimental model and its pathological and immunological reliability. Clinics, 75, e1858.

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Skeletal Muscle Fiber Morphometry

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Fragments from medial third of skeletal muscles (gastrocnemius, soleus and biceps brachii) were snap-frozen in liquid nitrogen and stored at − 80 °C. Semi-serial 10 μm-thick cross-sections were stained with hematoxylin and eosin. Skeletal muscle section images were captured at 20× magnification with a BX 50 microscope (Olympus, Tokyo, Japan) and the minor diameter of 100 muscle fibers (per animal) were analyzed using Image-Pro® Plus software version 4.5 (Media Cybernetics Inc., Rockville, MD, USA).

Lima Y.C., Kurauti M.A., da Fonseca Alves G., Ferezini J., Piovan S., Malta A., de Almeida F.L., Gomes R.M., de Freitas Mathias P.C., Milani P.G., da Costa S.C, & Mareze-Costa C.E. (2019). Whey protein sweetened with Stevia rebaudiana Bertoni (Bert.) increases mitochondrial biogenesis markers in the skeletal muscle of resistance-trained rats. Nutrition & Metabolism, 16, 65.

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Aortic Plaque Quantification via Oil-Red-O Staining

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Following cardiac puncture, the aorta was harvested, sampled, washed with distilled water, dipped in 78% methanol and stained for 40 min with 0.16% Oil-Red-O dissolved in 78% methanol/0.2 M NaOH (Merck KGaA) at room temperature. For morphological analysis, 7 μm frozen sections were stained with hematoxylin and eosin (H&E) according to standard protocols. For quantification of the lesion area, serial cross sections of the aortic roots (7 μm thick) and five serial sections were prepared from each location and conventionally stained with Oil-Red-O according to the method described by Yang
et al.
[17] (link). The lesion areas were quantified by a single observer blinded to the experimental protocol. The stained plaque areas were quantified using Image Pro Plus software version 4.5 (Media Cybernetics, Rockville, USA).

Ma S., Lu G., Zhang Q., Ding N., Jie Y., Zhang H., Xu L., Xie L., Yang X., Zhang H, & Jiang Y. (2022). Extracellular-superoxide dismutase DNA methylation promotes oxidative stress in homocysteine-induced atherosclerosis: EC-SOD DNA methylation regulates atherosclerosis in ApoE –/– mice. Acta Biochimica et Biophysica Sinica, 54(9), 1222-1233.

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Quantifying Cardiac Interstitial Collagen

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The hearts were sectioned in a cryostat (6 µm) and were stained with collagen-specific picrosirius red (PSR) for measurements of the content of interstitial collagen [28] (link). The images were obtained using a polarized light microscope [29] (link) (Olympus BX51) with a camera attached (Olympus DP72, 40X objective). For image acquisition and quantification, the image pro plus software version 4.5 (Media Cybernetics, Silver Spring, Maryland, USA) was used. For each animal, 40 fields were selected, with the chosen fields being located far from the infarcted area and from the pericardial region, in order to determine the percentage of collagen volume fraction (CVF). The collagen volume fraction was defined as the sum of all stained interstitial collagen tissue areas divided by the entire tissue area [30] (link).

Alves J.P., Nunes R.B., Stefani G.P, & Dal Lago P. (2014). Resistance Training Improves Hemodynamic Function, Collagen Deposition and Inflammatory Profiles: Experimental Model of Heart Failure. PLoS ONE, 9(10), e110317.

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Myocardial Infarction Model in Mice

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Mice were anesthetized (40 mg/kg pentobarbital–40 mg/kg ketamine) and subjected to 45-min left anterior descendent coronary artery ligation followed by 24-h reperfusion. Hearts were removed and retrogradely perfused with Krebs-Henseleit buffer for 1 min. The coronary artery was relegated at the same point, and the heart was perfused with 5% Evans blue and sliced in 6 short-axis sections (Zivic Instruments heart slicer matrix) that were incubated with 1% triphenyltetrazolium chloride (Sigma Chemical) for 10 min at 37 °C. The slices were weighed and photographed from both sides. The area at risk (Evans blue–negative) and the area of necrosis (Evans blue–negative and triphenyltetrazolium chloride–negative) were semiautomatically determined (Image ProPlus software version 4.5, Media Cybernetics) in the digital images and expressed as average percent area from both sides of each slice and corrected for slice weight: area at risk weight = (weight slice 1 × % area at risk slice 1) + … + (weight slice N × % area at risk slice N). Area of necrosis weight was calculated in the same manner. Finally, infarct size was calculated as percentage of the area at risk (28 (link)). To explore the effect of sGC activation on myocardial infarction, a subgroup of animals from the control and ID groups received 10 μg/kg ataciguat (HMR1766, Sigma-Aldrich) intraperitoneally 10 min before ischemia.

Inserte J., Barrabés J.A., Aluja D., Otaegui I., Bañeras J., Castellote L., Sánchez A., Rodríguez-Palomares J.F., Pineda V., Miró-Casas E., Milà L., Lidón R.M., Sambola A., Valente F., Rafecas A., Ruiz-Meana M., Rodríguez-Sinovas A., Benito B., Buera I., Delgado-Tomás S., Beneítez D, & Ferreira-González I. (2021). Implications of Iron Deficiency in STEMI Patients and in a Murine Model of Myocardial Infarction. JACC: Basic to Translational Science, 6(7), 567-580.

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Measuring Vaginal Epithelial Thickness

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Two vaginal 5mm biopsies were performed by placing the animals in ventral recumbency and taking biopsies at the 12 and either the 3 or 9 o'clock position at both day 0 (peak luteal) and day 14 (peak follicular) phase of the cycle.
The vaginal epithelial thickness was measured using ImageProPlus software, version 4.5 (Media Cybernetics, Silver Springs, MD, USA) on appropriately oriented H&E stained sections using a Leica microscope (Leica Microsystems Inc., Bannockbum, IL, USA) interfaced to a digital camera (Spot Insight color camera; Diagnostic Instruments Inc., Sterling Heights, MI, USA). The software was calibrated for each objective using a stage micrometer. An average of 16 images were collected from each section taken at 100x magnification. For each image, ten measurements were taken by applying an electronic grid to eliminate observer bias, and measurements were taken from the point where each gridline intersected the basal epithelium using ImageProPlus software (Fig. 1A).

Hadzic S.V., Wang X., Dufour J., Doyle L., Marx P.A., Lackner A.A., Paulsen D.B, & Veazey R.S. (2014). Comparison of the vaginal environment of Macaca mulatta and Macaca nemestrina throughout the menstrual cycle. American journal of reproductive immunology (New York, N.Y. : 1989), 71(4), 322-329.

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FFPE Tissue Immunostaining and Quantification

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FFPE samples were obtained and fixed, after that, they underwent staining with hematoxylin and eosin-H&E (Harris Hematoxylin: NewProv, Cod. PA203, Pinhais, Brazil; Eosin: BIOTEC Reagentes Analíticos, Cod. 4371, Pinhais, Brazil). Subsequently, specific staining for TLR4; ACE2; IL-1β; IL-18; NF-κB; ASC; NLRP-3 (or NALP); CASP1; CASP9; GDSM-D; NOX4; TNF-α was performed, and the slides were then scanned using Axio Scan.Z1 Scanner (ZEISS, Jena, Germany), and then ZEN Blue Edition (ZEISS, Jena, Germany) was utilized to randomly generate high-power fields (HPF = 40× objective). Images were randomly generated by the software, with no investigator’s interference. The immunopositivity areas were measured by the Image-Pro Plus software version 4.5 (Media Cybernetics, Rockville, MD, USA). Subsequently, these stained areas were converted into percentages per total tissue area to enable statistical analysis. This study analyzed overall marker expression. Staining information can be found on supplementary Table S1

Baena Carstens L., Campos D’amico R., Fernandes de Moura K., Morais de Castro E., Centenaro F., Silva Barbosa G., Vieira Cavalcante da Silva G., Brenny I., Honório D’Agostini J.C., Hlatchuk E.C., Pissette de Lima S., Camargo Martins A.P., De Castro Deus M., Konzen Klein C., Kubaski Benevides A.P., Nagashima S., Machado-Souza C., Pinho R.A., Pellegrino Baena C, & de Noronha L. (2022). Lung Inflammasome Activation in SARS-CoV-2 Post-Mortem Biopsies. International Journal of Molecular Sciences, 23(21), 13033.

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Immunohistochemical Analysis of Renal Proteins

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Renal tissue sections (4 µm) were subjected to immunohistochemical staining based on the published method with specific antibodies anti-RAGE (1:100, ab37647; Abcam), anti-nephrin (1:500, ab58968; Abcam), and anti-podocin (1:400, H-130, sc-21009; Santa Cruz Biotechnology, Inc.) (25 (link)). Color was developed by incubating with diaminobenzidine and counterstaining with hematoxylin. Controls were obtained by replacing the primary antibody with PBS. Semi quantitative analysis of the percentage of positive staining area in the glomeruli and tubules was evaluated using a computer imaging analysis system (Image-Pro Plus software version 4·5; Media Cybernetics, Inc., Rockville, MD, USA). The brown areas were judged as positive.

Li X., Gao Z., Gao H., Li B., Peng T., Jiang B., Yang X, & Hu Z. (2017). Nephrin loss is reduced by grape seed proanthocyanidins in the experimental diabetic nephropathy rat model. Molecular Medicine Reports, 16(6), 9393-9400.

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Immunohistochemical Analysis of Lung Biopsy

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Minimally invasive lung biopsy was performed through a left anterior mini thoracotomy with upper left lobe lingular segment resection. The resected pieces were 3 × 3 cm. The samples were formalin-fixed, paraffin embedded (FFPE) and stained with hematoxylin and eosin (H&E) to find the properly areas for the IHC techniques.
The FFPE lung samples were also performed by IHC reactions using primary monoclonal antibodies anti IL-6 (Monoclonal/Mouse, Ab9324, 1:400, Abcam), TNFα (Policlonal/Rabbit, Ab6671, 1:100, Abcam), CD163 (Policlonal/Rabbit, 14215, 1:1000, Thermo Fisher), FCN3 (Policlonal/Rabbit, Orb30580, 1:600, Biorbyt), and MBL (Policlonal/Rabbit, PAB480Hu02, Cloud-Clone, 1:200).
The slides of IL-6, TNFα, CD117, FCN3, and MBL were scanned on the Axio Scan Scanner Z1 (Carl Zeiss, Germany). Afterward, 10 high-power field (HPF) were selected of each sample and the positive areas were quantified using Image-Pro Plus software version 4.5 (Media Cybernetics, USA). Subsequently, these areas were converted into percentage to enable statistical analysis.
CD163 slides were also used to score macrophages, in 30 HPF. The HPF was chosen randomly from the septum and lumen alveolar, where the macrophages were scored.

Malaquias M.A., Gadotti A.C., Motta-Junior J.D., Martins A.P., Azevedo M.L., Benevides A.P., Cézar-Neto P., Panini do Carmo L.A., Zeni R.C., Raboni S.M., Fonseca A.S., Machado-Souza C., Moreno-Amaral A.N, & de Noronha L. (2020). The role of the lectin pathway of the complement system in SARS-CoV-2 lung injury. Translational Research, 231, 55-63.

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Fluorescence and Bright Field Microscopy Imaging Analysis

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Fluorescence images were taken with an Eclipse TE 2000-S microscope equipped with a DS-Qi2 digital SLR camera (Nikon Instruments Inc., Tokyo, Japan, No. 750549) A light microscope (Leica Mycrosystems, Shinjuku City, Tokyo, Japan, No. 512834) equipped with a motorized stage, focus control system, and a color digital camera (Coolsnap-Pro, Media Cybernetics, Rockville, MD, USA, No. A00M82009) were used for bright field images. Measurements were carried out using the Image Pro Plus software version 4.5 (Media Cybernetics, Silver Spring, MD, USA) by investigators blind to the animal’s genotype.

Mottolese N., Uguagliati B., Tassinari M., Cerchier C.B., Loi M., Candini G., Rimondini R., Medici G., Trazzi S, & Ciani E. (2023). Voluntary Running Improves Behavioral and Structural Abnormalities in a Mouse Model of CDKL5 Deficiency Disorder. Biomolecules, 13(9), 1396.

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