To determine the subcellular distribution of mitochondria, the cells were treated with 50 nM MitoTracker™ Red (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 30 min to stain the mitochondria and cell cytosol. The mean mitochondrial length was determined by measuring 10 individual mitochondria from cells obtained by fluorescence microscopy using the cellSens software version 1.16 (Olympus Corporation).
Cellsens software version 1
Manufactured by Olympus
Sourced in Japan, Australia
CellSens software version 1.6 is a digital imaging and analysis platform for microscopy applications. The software provides tools for image acquisition, processing, and measurement. It is designed to work with Olympus microscopy hardware.
Automatically generated - may contain errors
Lab products found in correlation
Mitotracker red, by Thermo Fisher Scientific (1 mentions)
Ix83 inverted fluorescent microscope, by Olympus (1 mentions)
Prism 7, by GraphPad (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Jc 1 mitochondrial membrane potential assay kit, by Cayman Chemical (1 mentions)
3 amino 9 ethylcarbazole aec, by Vector Laboratories (1 mentions)
Ix73 microscope, by Olympus (1 mentions)
Erbb2, by Abcam (1 mentions)
Prism 9, by GraphPad (1 mentions)
Picro sirius red solution, by ScyTek Laboratories (1 mentions)
Bz x700 microscope, by Keyence (1 mentions)
Mounting medium, by Muto Pure Chemicals (1 mentions)
Rat anti mouse f4 80, by Bio-Rad (1 mentions)
Rat anti mouse ly6g, by Abcam (1 mentions)
Goat anti collagen 4, by Southern Biotech (1 mentions)
10 protocols using cellsens software version 1
1
Quantifying Mitochondrial Distribution
2
Quantifying DNA-Nanocarrier Cell Clusters
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The RCA reactions proceeded for 0.5, 1, 1.5, 2 or 2.5 h, respectively, forming DNC–Cell clusters in different sizes. The fluorescent indicator Cy3-C-strands were also used to show the DNA network between cells. Cells were washed with PBS twice immediately, followed by resuspending in PBS. Cells were seeded on a 12-well plate then imaged by Fluorescent Inverted Microscope (IX83, Olympus, Tokyo, Japan). The DNC–Cell clusters were measured and counted by cellSens software version 1.16, (Olympus, Tokyo, Japan), using a measurement function. The size means the average diameter of the clusters and was fitted by Gaussian fitting using GraphPad Prism 7, software (San Diego, CA, USA). The percentage of DNC–Cell clusters is calculated by the number of cells that formed clusters dividing the number of total cells.
3
Femur Histomorphometric Analysis
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All right femurs were put in a 19% EDTA solution for 14 days, and the solution was changed daily. Subsequently, all samples were sent to the Translational Pathology Core Laboratory (TPCL) at the UCLA Department of Pathology for paraffin embedding. Longitudinal sections of 5 μm thickness were created by microtome. All slides were used for hematoxylin and eosin (H&E), trichrome, tartrate-resistant acid phosphatase (TRAP), and osteocalcin (OCN) staining. All specimens were analyzed under an Olympus BX51 microscope (Olympus Corp.) using cellSens software version 1.6 (Olympus Corp.). Six consecutive images at the distal femur region were acquired for OCN and TRAP analyses, which were completed by three blinded examiners using ImageJ software v1.48 (National Institutes of Health). Parameters of osteocalcin+bone-lining cells per bone perimeter (OCN+cells/Bpm, mm−1) and surface (Ob.S/Bs, %), TRAP+bone-lining cells per bone perimeter (TRAP+cells/Bpm, mm−1), and surface (Oc.S/Bs, %) were used as previously reported.12 (link)
4
Mitochondrial Membrane Potential Assay
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The cells were incubated at 37 °C or 43 °C for 1 h in the presence of 0.1% 2,5-HD and then maintained at 37 °C for 24 h. To observe the polarization of the mitochondrial membrane, the JC-1 mitochondrial membrane potential assay kit (Cayman Chemical, Ann Arbor, MI, USA) was used according to the manufacturer’s instructions. The fluorescence images of JC-1 aggregates [red] and monomers [green] were examined using a fluorescence microscope (IX-51). The fluorescence intensities were calculated using CellSens software version 1.6 (Olympus, Tokyo, Japan).
5
Quantifying Calvarial Bone Volume Using IHC
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Samples were fixed in 4% PFA for 24 h and decalcified in 10% ethylenediaminetetraacetic acid (0.1 mol·L-1, pH 7.1) solution for 2 weeks. After decalcification, the parietal bone was removed from the cranium and embedded in paraffin. Tissue samples were sectioned into 5-μm sections and stained with hematoxylin and eosin at the University of California, Los Angeles, Tissue Procurement Core Lab. Slides were submerged in 0.1% trypsin diluted in 1X PBS for 10–20 min at 37 °C for antigen retrieval and then incubated with anti-KDM6B antibody (1:100, Abcam, catalog no. ab85392) as a primary antibody. Tissue sections were visualized using 3-amino-9-ethylcarbazole (AEC; Vector Laboratories), followed by counterstaining with hematoxylin. Sections were sealed with Faramount aqueous mounting solution (Dako) as previously described.68 (link)An Olympus IX73 microscope and cellSens software version 1.6 (Olympus Corporation) were used to visualize and quantify the bone volume of calvarial defects in IHC. Three random fields of view per section sample were randomly taken for each specimen, and the average values were used as the data point for each specimen.
6
Immunohistochemical Analysis of Mouse Mammary Glands
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Mammary glands and breast tumors were dissected from mice, fixed in 10% neutral formaldehyde for 16 hours at room temperature, gradually dehydrated in ethanol, and embedded in paraffin. Paraffin sections (2 mm thick) were cut, deparaffinized with xylene, and subjected to hematoxylin and eosin staining using the standard protocol. For immunohistochemical staining, the tissue sections were subjected to permeabilization in 0.2% Triton X-100 in Tris-buffered saline for 10 minutes, optimal antigen retrieval using 10 mmol/L citric acid buffer (pH 6.0) for 20 minutes at 98 C, and incubation with antibodies recognizing ERa (1 mg/mL; Santa Cruz Biotechnology, Dallas, TX), Ki-67 (5 mg/mL; BioLegend, San Diego, CA), or ErbB2 (10 mg/mL; Abcam, Cambridge, UK) for 1 hour at room temperature. They were subsequently incubated with horseradish peroxidaseeconjugated polymerized secondary antibodies recognizing rabbit or mouse IgG (Dako, Glostrup, Denmark), and visualized using diaminobenzidine (Dako). Proliferation of mammary glands was quantified by measuring the mammary epithelial cell areas using the cellSens software version 1.6 (Olympus, Tokyo, Japan) equipped in a light microscope BX63 (Olympus). The number of ERa-and Ki-67epositive cells was counted manually under the microscope and expressed as values against the whole cell number per unit area.
7
Quantifying Apoptosis and Immune Cells in Kidney Injury
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Cell death was visualized by TUNEL staining on 4 μm sections of formalin-fixed tissues using the ApopTag Peroxidase In Situ Apoptosis Detection Kit (Millipore-Chemicon, Ryde, NSW, Australia). The number of TUNEL+ cells were counted under high power (×400) in the outer medulla (IRI model) or in the entire cortex (UUO model).
Immunoperoxidase staining for neutrophils in the IRI model was performed on 5 μm sections of 2% paraformaldehyde-fixed, cryostat tissue sections as previously described [45 (link)]. The number of Ly6G+ neutrophils was counted in high power fields (×400) in the outer medulla. Immunostaining for macrophages, α-SMA, collagen IV, and CD31 in the UUO model was performed on 4 µm sections of methylcarn-fixed, paraffin-embedded tissue. The area of interstitial staining for F4/80+ macrophages, α-SMA, collagen IV, and CD31 was determined in the entire cortex (excluding large vessels) under medium power (×200) by image analysis using cellSens software version 1.18 (Olympus Australia, Notting Hill, Victoria, Australia).
Immunoperoxidase staining for neutrophils in the IRI model was performed on 5 μm sections of 2% paraformaldehyde-fixed, cryostat tissue sections as previously described [45 (link)]. The number of Ly6G+ neutrophils was counted in high power fields (×400) in the outer medulla. Immunostaining for macrophages, α-SMA, collagen IV, and CD31 in the UUO model was performed on 4 µm sections of methylcarn-fixed, paraffin-embedded tissue. The area of interstitial staining for F4/80+ macrophages, α-SMA, collagen IV, and CD31 was determined in the entire cortex (excluding large vessels) under medium power (×200) by image analysis using cellSens software version 1.18 (Olympus Australia, Notting Hill, Victoria, Australia).
8
Electrophysiology and Immunostaining Analysis
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For electrophysiology experiments, data were expressed as the mean ± standard error of mean. Results were analyzed using non-parametric Wilcoxon matched-pairs signed rank tests or two-way ANOVA. For immunostaining experiments, images were analyzed using Olympus cellSens software version 1.18. Mean gray intensity values for each region of interest were normalized to the control group at each timepoint and plotted. Two-way ANOVA with Bonferroni multiple comparisons was performed to assess differences between groups. P < 0.05 was considered statistically significant. Statistical tests were done using Prism 9.5.1 software program (GraphPad Software).
9
Heart Collagen Fiber Analysis Protocol
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The heart sections were immersed in Maeda’s resorcinol fuchsin stain solution (Muto Pure Chemicals Co., Ltd.) for 1 h and washed with 100% ethanol. Heart sections (5-μm thick) were mounted on glass slides and immersed in Weigert’s iron hematoxylin solution and washed with Milli-Q water. The specimens were then immersed in Picro–Sirius Red solution (ScyTek Laboratories, Inc., West Logan, UT, USA) for 15 min, air-dried, and rinsed with xylene. After staining, the tissue sections were dehydrated in 70%, 95%, and 100% ethanol solutions and ethanol/xylene mixed solution, immersed in xylene four times, and sealed using a mounting medium (Muto Pure Chemicals Co., Ltd., Tokyo, Japan). The sections were observed under a bright-field microscope BX53, (Olympus, Tokyo, Japan) using the cellSens software version 1.18 (Olympus) and BZ-X700 microscope (Keyence, Osaka, Japan). The collagen fiber content in the compact layer and cardiomyocyte density were measured using the Fiji software version 2.3.0 [75 (link)].
10
Immunostaining and Cell Death Analysis
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Immunoperoxidase staining for macrophages (rat anti-mouse F4/80; Bio-Rad, Gladesville, Australia) and collagen IV (goat anti-collagen IV; Southern Biotechnology, Birmingham, AL, USA) was performed on 4 µm sections of methylcarn-fixed tissue as previously described [33 (link)]. Immunoperoxidase staining for neutrophils (rat anti-mouse Ly6G; Abcam, Melbourne, Australia) and T cells (rat anti-mouse CD3; Bio-Rad) was performed on 5 µm cryostat sections of tissues fixed in 2% paraformaldehyde as previously described [24 (link)].
In the IRI model, the number of neutrophils was counted in high-power fields (400×) covering the entire inner cortex and outer medulla. In the UUO model, the number of macrophages, T cells and neutrophils was counted in high-power fields (400×) covering the entire cortex. The area of interstitial collagen IV staining in the entire cortex (excluding large vessels) was assessed under medium power (200×) by image analysis using cellSens software version 1.18 (Olympus Australia, Notting Hill, Australia).
Cell death was assessed in 4 μm sections of formalin-fixed tissue by TUNEL staining with the ApopTag Peroxidase In Situ Apoptosis Detection Kit (Millipore-Chemicon, Ryde, Australia). The number of TUNEL+ tubular cells in the inner cortex and outer medulla were counted in high-power (400×) fields. All scoring was performed on blinded slides.
In the IRI model, the number of neutrophils was counted in high-power fields (400×) covering the entire inner cortex and outer medulla. In the UUO model, the number of macrophages, T cells and neutrophils was counted in high-power fields (400×) covering the entire cortex. The area of interstitial collagen IV staining in the entire cortex (excluding large vessels) was assessed under medium power (200×) by image analysis using cellSens software version 1.18 (Olympus Australia, Notting Hill, Australia).
Cell death was assessed in 4 μm sections of formalin-fixed tissue by TUNEL staining with the ApopTag Peroxidase In Situ Apoptosis Detection Kit (Millipore-Chemicon, Ryde, Australia). The number of TUNEL+ tubular cells in the inner cortex and outer medulla were counted in high-power (400×) fields. All scoring was performed on blinded slides.
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