Immunohistochemistry was used to detect the expression of sirt3 protein in the renal allograft tissue. Antigens were retrieved using the citric acid buffer (pH 6.0) microwave antigen retrieval method and detected via PV-9001 staining (Golden Bridge Biotechnology Co., Beijing, China). Tissues were boiled for 15 min at a temperature ranging 95–100°C and staining with PV-9001 was performed at room temperature for 60 min. The paraffin sections 4 µm thick were hydrated in an alcohol gradient (75, 85, 95, and 100% at 37°C for 3 min per concentration) and 3% hydrogen peroxide was used to block the endogenous peroxidase. Sections were then incubated with rabbit polyclonal anti-sirt3 antibodies (cat. no. 2627, 1:100; Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4°C. Sections were washed with PBS, incubated with the goat anti-rabbit secondary antibody (cat. no. PV-9001, diluted 1:100; Golden Bridge Biotechnology Co., Ltd., Beijing, China) for 20 min at room temperature and washed a further three times with PBS. Sections were then incubated in a 3,3′-diaminobenzidine chromogen solution (OriGene Technologies, Inc., Rockville, MD, USA) and re-stained with hematoxylin at 37°C for 1 min. The intensity of positive staining for sirt3 was quantified using Image-Pro Plus software version 7.0.1.658, Media Cybernetics, Inc. (Rockville, MD, USA).
Image pro plus software version 7
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Image-Pro Plus software, version 7.0, is a powerful image analysis and processing application. It provides tools for capturing, processing, analyzing, and managing digital images. The software supports a wide range of image file formats and offers a comprehensive set of features for image enhancement, measurement, and quantification.
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Lab products found in correlation
Wga alexa488, by Thermo Fisher Scientific (2 mentions)
Buffered formalin acetate, by Thermo Fisher Scientific (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Cell d software, by Olympus (1 mentions)
Bx41tf, by Olympus (1 mentions)
Eclipse e600 microscope, by Nikon (1 mentions)
Dfc7000 t camera, by Leica (1 mentions)
Dmi6000 inverted microscope, by Leica (1 mentions)
Dfc425, by Leica (1 mentions)
Tcs sp8 confocal microscope, by Leica (1 mentions)
Fl 145, by Santa Cruz Biotechnology (1 mentions)
Tcs sp8, by Leica (1 mentions)
Fluoromount g, by Thermo Fisher Scientific (1 mentions)
Click it edu alexa fluor 488 imaging kit, by Thermo Fisher Scientific (1 mentions)
Ecl reagent, by Beyotime (1 mentions)
Bca assay, by Solarbio (1 mentions)
Hrp conjugated igg secondary antibody, by Abcam (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Eclipse te2000 e, by Nikon (1 mentions)
19 protocols using image pro plus software version 7
1
Sirt3 Expression in Renal Allograft Tissue
2
Lung Histology and Imaging Procedure
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After euthanasia, lungs were perfused with ice-cold PBS via the right ventricle of the heart. Using 10% buffered formalin acetate (Fisher Scientific), the lungs were inflated to a pressure of 25 cm H2O and fixed overnight. Subsequently, lungs were embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin–eosin (H&E), periodic acid–Schiff (PAS), or mucicarmine reagents at the Histology Facility of the Goodman Cancer Research Centre (McGill University). Representative photographs of lung sections were taken using a BX51 microscope (Olympus), QICAM Fast 1394 digital charge-coupled device camera (QImaging), and Image-Pro Plus software version 7.0.1.658 (Media Cybernetics).
3
Quantitative Immunohistochemical Analysis
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All quantitative analyses of immunohistochemistry were performed with the Image Pro Plus software version 7 (Media Cybernetics Europe, Marlow, UK) by blinded investigators. Digital images of the immunostainings were captured with a light microscope (BX41TF, Olympus) using the Cell D software (Olympus). Images of 20× magnification, covering the complete nerve biopsy were quantified. The surface area stained is expressed as percentage of total area examined. For the mouse nerves error bars represent the standard deviation and for the human nerves error bars indicate standard error of the mean.
4
Histological Analysis of Liver Tissue
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The liver tissues fragments from necropsies were fixed in 10% formaldehyde, pH = 7.2, and processed and blocked-in paraffin resin. Sections of 5 μm thickness were made in a microtome (American Optical, Spencer model), and mounted in glass slides. Before staining, the slides were deparaffinized in three baths of xylene and rehydrated with decreasing concentrations of ethanol (100 to 70%). Then, sections were submitted to standard and special staining with Hematoxylin and Eosin (H.E.), Periodic Acid Schiff (PAS), Picro Sirius Red and prepared for visualization under a Nikon ECLIPSE E600 microscope. Photomicrographs were captured using Image-Pro Plus software version 7 (Media Cybernetics).
5
Adhesion and Migration Assays for Immune Cells
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For the adhesion assays, HUVECs were cultured in 6-well plates until 85–90% confluent. THP-1 cells were labeled with calcein-AM (2.5 μM) for 30 min at room temperature and washed once with PBS. Calcein-AM-labeled THP-1 cells (0.5 × 106/mL) were incubated with the HUVEC monolayer for 3 h at 37 °C. The wells were washed twice with PBS to remove unbound or loosely bound THP-1 cells, and luminescence quantified.
A trans-well migration assay was performed in 24-well, trans-well inserts with 5 μm pores (Corning). THP-1 cells were labeled with calcein-AM, and 1 × 106 cells in serum-free RPMI media were added to the trans-well upper chambers. CM from MCF-7 or MDA-MB-231 cells was added to the lower chambers as chemoattractant. After 6 h at 37 °C, the inserts were washed three times with PBS and non-migrating cells removed from the upper chambers by scrubbing with a cotton swab. Migrating cells were imaged at 10X using a Leica DMI6000 inverted microscope with a Leica DFC7000T camera (Leica Microsystems, GmbH, Wetzlar, Germany) and quantified using ImagePro Plus software version 7 (Media Cybernetics, Inc., Rockville, MD, USA).
A trans-well migration assay was performed in 24-well, trans-well inserts with 5 μm pores (Corning). THP-1 cells were labeled with calcein-AM, and 1 × 106 cells in serum-free RPMI media were added to the trans-well upper chambers. CM from MCF-7 or MDA-MB-231 cells was added to the lower chambers as chemoattractant. After 6 h at 37 °C, the inserts were washed three times with PBS and non-migrating cells removed from the upper chambers by scrubbing with a cotton swab. Migrating cells were imaged at 10X using a Leica DMI6000 inverted microscope with a Leica DFC7000T camera (Leica Microsystems, GmbH, Wetzlar, Germany) and quantified using ImagePro Plus software version 7 (Media Cybernetics, Inc., Rockville, MD, USA).
6
Daphnia magna Reproduction Assay
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Survival and the number of neonates produced were assessed three times per week, prior to each water change. Female and male neonates were enumerated by removing all neonates from exposure chambers, growing neonates in 2‐L plastic beakers at the prescribed feeding ration for 5–7 days and identifying the sex under a dissecting microscope according to available literature and keys (Ebert, 2005 ; Olmstead & Leblanc, 2006 ). Carapace length was determined at test termination by capturing calibrated D. magna images from at least five individuals per replicate using a digital camera (Leica Microsystems Ltd., DFC425) attached to a dissection microscope (S6 E, Leica Microsystems Ltd.). Length was measured from the eyespot to the base of the anal spine using image‐pro plus software version 7 (Media Cybernetics, Inc.).
7
Immunohistochemical Analysis of Extracellular Matrix
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Each paraffin-embedded section was deparaffinized, dehydrated, and then boiled under high pressure and temperature in 0.01 mol/L sodium citrate for 3 min. Next, the sections were treated with 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity. The sections were then incubated with the primary antibody (anti-collagen-1, 1:100, no. 2035; anti-collagen-2, 1:100, no. 2036; anti-TGF-β, 1:200, no. 2042; anti-MMP-1, 1:100, no. 2038; anti-MMP-3, 1:100, no. 2040, Bosheng Corp., Beijing, China) for 60 min at 37 °C. Then, the sections were incubated with an appropriate mouse-anti-goat secondary antibody (Dako Corp., Copenhagen, Denmark) at 37 °C for 20 min and colored with diaminobenzidine reagent (Dako Corp.). Finally, the immunohistochemical sections were counterstained with hematoxylin and observed under a light microscope. Images were saved using Image-Pro Plus software, version 7.0 (Media Cybernetics, Rockville, MD, USA). The mean grey values were used as indicators of the positive expression of collagen-1, collagen-2, and TGF-β, and the positive-expression rate were utilized as indicators of positively-stained MMPs. Visual fields of each image were randomly selected for further analysis.
8
Histological Analysis of Cardiac Remodeling
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After the completion of the experiments, the animals were euthanized. The hearts were harvested and arrested in diastole with 0.5% KCl (w/v) in PBS. The heart weights (HW) were determined and normalized to the body weight (BW). Then, part of each heart was immersed in 4% paraformaldehyde perfusion at 4 °C overnight. On the next day, the tissue samples were transferred to 70–100% ethanol for dehydration before embedding in paraffin. Heart sections (5 µm intervals) were deparaffinized and stained with hematoxylin and eosin (H&E), picrosirius red staining (Sigma-Aldrich) according to routine procedures. Images were acquired using Nikon Eclipse Ni light microscopy.
To determine the myocyte cross-sectional area, heart sections were incubated with wheat germ agglutinin-Alexa488 (WGA-Alexa488, Thermo Fisher Scientific, W11261) for 1 h to visualize the membranes and with 4,6 diamidino-2-phenylindole (DAPI) for 20 min to observe the nuclei. At least 100 randomly selected myocytes were measured from five animals/groups, and the average values were used for analysis. All measurements were made using ImagePro Plus software version 7.0 (Media Cybernetics, Rockville, MD). All morphometric analyses were performed in a blinded fashion. Sections were measured using a Leica TCS SP8 Confocal microscope (Leica, Wetzlar, Germany). All histopathologic data were confirmed by a blinded pathologist.
To determine the myocyte cross-sectional area, heart sections were incubated with wheat germ agglutinin-Alexa488 (WGA-Alexa488, Thermo Fisher Scientific, W11261) for 1 h to visualize the membranes and with 4,6 diamidino-2-phenylindole (DAPI) for 20 min to observe the nuclei. At least 100 randomly selected myocytes were measured from five animals/groups, and the average values were used for analysis. All measurements were made using ImagePro Plus software version 7.0 (Media Cybernetics, Rockville, MD). All morphometric analyses were performed in a blinded fashion. Sections were measured using a Leica TCS SP8 Confocal microscope (Leica, Wetzlar, Germany). All histopathologic data were confirmed by a blinded pathologist.
9
PCNA Expression in Cell Lines
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The expression of PCNA in HaCaT, HUVEC, and NIH/3T3 cells was detected by indirect immunofluorescent assay. The cells were seeded in six-well plates at a density of 1 × 104 cells/well, in 1.5 mL of DMEM medium supplemented with 5% FBS and antibiotic–antimycotic solution at 37 °C, under a humidified atmosphere of 5% CO2 and 95% air.
After 24 h of incubation, the DMEM medium was replaced with fresh DMEM and the treatments—calreticulin (2.5, 5, 7, 10 and 15 pg), AuNPs (1.5, 3, 6, and 15 µM) or AuNPs-calreticulin (1.5, 3, 6, and 15 µM)—were added to the seeded cells. After incubation for three days, the cells were permeabilized with Triton 0.1% and 1% sodium citrate in PBS for 2 min and washed three times with PBS. Then, the nonspecific binding sites were blocked with 20% FBS in PBS for 30 min and incubated for 18 h with anti-PCNA antibody and diluted 1:50 in PBS. The presence antibodies were detected with anti-mouse IgG2a FITC, and cells were counterstained with DAPI for 30 min. Finally, cells were washed with methanol and methanol-PBS, and were photographed at 40× in a confocal microscope (Olympus X70). The images were analyzed using Image-Pro Plus software, version 7.0. (Media Cybernetics, Rockville, MD, United States), and the pixels were correlated with the intensity of the signal.
After 24 h of incubation, the DMEM medium was replaced with fresh DMEM and the treatments—calreticulin (2.5, 5, 7, 10 and 15 pg), AuNPs (1.5, 3, 6, and 15 µM) or AuNPs-calreticulin (1.5, 3, 6, and 15 µM)—were added to the seeded cells. After incubation for three days, the cells were permeabilized with Triton 0.1% and 1% sodium citrate in PBS for 2 min and washed three times with PBS. Then, the nonspecific binding sites were blocked with 20% FBS in PBS for 30 min and incubated for 18 h with anti-PCNA antibody and diluted 1:50 in PBS. The presence antibodies were detected with anti-mouse IgG2a FITC, and cells were counterstained with DAPI for 30 min. Finally, cells were washed with methanol and methanol-PBS, and were photographed at 40× in a confocal microscope (Olympus X70). The images were analyzed using Image-Pro Plus software, version 7.0. (Media Cybernetics, Rockville, MD, United States), and the pixels were correlated with the intensity of the signal.
10
Mitochondrial Structure Visualization and Quantification
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Mitochondria were stained using immunocytochemistry (ICC) (as described above) with primary antibodies against TOM20 (FL145 Santa Cruz Biotech, Dallas, Texas; 1:100); and ATPB (AB5452 Abcam, Cambridge, UK; 1:500). Imaging was performed by confocal microscopy (Leica TCS S5 microscope, Leica microsystems, Wetzlar, Germany). Image processing, three-dimensional (3D)-modeling and quantitative analysis of mitochondrial structures were performed using the Image-Pro Plus software (version 7.0) (Media Cybernetics), as described previously [32 (link), 36 (link)]. Further details are provided in Additional file 1 : Supplemental methods.
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