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Imagej software

Manufactured by National Instruments
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ImageJ is an open-source image processing software designed for scientific and analytical use. It provides a wide range of tools for image acquisition, analysis, and processing. The core function of ImageJ is to enable users to view, edit, analyze, process, save, and print 8-bit, 16-bit, and 32-bit images. It supports a variety of image file formats and provides a customizable user interface for efficient image manipulation and data extraction.

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13 protocols using imagej software

1

Western Blot Analysis of Megalin in Mouse Kidneys

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Total cellular extracts from mouse kidneys were prepared, and Western blotting was performed using a denaturing 3–8% Tris-acetate gel (Invitrogen, Carlsbad, CA, USA) under reducing conditions, as previously described [25 (link)]. Membranes were blocked and incubated with a primary rabbit anti-megalin antibody (kindly provided by Prof. Akihiko Saito, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan) [26 (link)] for 2 h at room temperature, followed by an incubation with horseradish peroxidase-conjugated secondary antibodies for 1 h. For loading controls, the same membranes were stripped and reprobed with a β-actin antibody (Sigma-Aldrich, St. Louis, MO, USA). Immunoreactive proteins were detected using an enhanced chemiluminescence (ECL) system according to the manufacturer's instructions (Amersham). Immunoblots were quantified using National Institutes of Health ImageJ software. The ratio between the intensities of megalin and β-actin bands was used to normalize megalin expression in each sample.
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2

Cardiac Histological Analysis: A Comprehensive Approach

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Sections of hearts samples with 5 μm thick sections fixed in 4% PFA and embedded in paraffin were stained with Masson's trichrome (KeyGen, China) to detect the degree of collagen deposition, or hematoxylin and eosin (H&E, KeyGen, China) to measure cardiomyocytes cross-sectional areas, or CD31 (1:100, Cell Signaling Technology, USA) immunohistochemical staining to measure myocardial capillary density. Images of the left ventricular area of each section were obtained by microscope (Nikon, Japan). Frozen sections of hearts in O.C.T Compound (optimal cutting temperature compound, Sakura, USA) were cut at 5 μm per section and stained with wheat germ agglutinin (WGA, Sigma, USA) to measure cardiomyocytes cross-sectional areas. Fluorescence images were obtained using a Zeiss confocal microscope (Carl Zeiss, Germany). Image J Software (National Institutes of Health) was used to quantify fibrotic region and measure cardiomyocytes cross-sectional areas in each section. The percentage of fibrosis was measured as fibrosis areas/total left ventricular areas x 100%.
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3

Lipid Droplet Imaging in Fly Larvae

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Ten larvae from each fly strain were infected with 10 S. carpocapsae symbiotic or axenic nematodes and samples were collected 24 h post-infection. Dissections were performed in 1X PBS, and fat body tissues were separated from the rest of the larval carcass. They were then fixed in 4% paraformalydehyde prepared in PBS for 30 min at room temperature followed by rinsing with 1X PBS twice. They were then incubated in the dark for 30 min in 1:1000 dilution of 0.05% Nile Red in 1 mg/ml of methanol. These tissues were then mounted in ProLongTM Diamond AntiFade Mountant with DAPI (Life Technologies). Images were obtained using a Confocal Olympus FluoViewTM FV1000 imaging system. Data were collected from fat body tissues of each of the 10 larvae. LD area was assessed using ImageJ software (National Institutes of Health). A minimum of five random regions were selected for LD size quantification from each fat body tissue.
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4

Chondrocyte Viability in Hydrogel Constructs

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The viability of chondrocytes in GelMA and GelMA-GC hydrogels was determined on days 1 and 28 of free-swelling and day 14 of intermittent uniaxial loading (total 28 days of culture) using a live/dead assay described elsewhere.97 (link) Briefly, after the push-out test, hydrogel constructs were sliced into two by a sterile scalpel, washed with PBS, and then incubated for 3 min at room temperature in PBS solution containing fluorescein diacetate (FDA; 1 μg/ml) and propidium iodide (PI; 1 μg/ml) (both from Sigma-Aldrich, USA). The samples were rewashed with PBS, and four Z-stack images per sample were acquired using a Zeiss Axio microscope (Carl Zeiss Axio Imager M2, GmbH, Germany) at each time point for each group (n = 4) and analyzed using Image J software (National Instruments, USA). Cell viability reported as a percentage of the total number of cells that were alive. Whole-slice images of hydrogels were also captured to represent an overall view of the impact of uniaxial loading on the top and bottom parts of hydrogel in cartilage-hydrogel constructs. To comprehensively examine the integration and cell viability of distinct cartilage-hydrogel areas, whole cartilage-hydrogel constructs were imaged on day 28 of culture with a Zeiss Axio microscope following a live/dead assay.
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5

Viability Assessment of oBMSC on Scaffolds

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The viability of oBMSC on scaffolds and coverslips was analysed on days 7 and 21 using double staining with ethidium homodimer-1 and calcein-AM in PBS (4 and 2 µM, respectively) (Merck Life Science, Gillingham, UK). Briefly, the scaffolds and coverslips were moved to a new plate and washed, once, with PBS. Subsequently, 500 µL of working solution was added to each well, and the plates were incubated for 1 h (5% CO2 and 37 °C). Afterward, the samples were gently rinsed twice with PBS and imaged with a confocal laser microscope (LSM 710, Zeiss, Oberkochen, Germany) at 488 and 543 nm. Cell viability was quantified using ImageJ software (National Instruments, Austin, TX, USA). Cells were manually counted using the multi-point tool. The percentage of viable cells were calculated according to the following equation: Live cellsTotal number of cells×100.
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6

Galectin-3 Immunofluorescence in Cell Lines and Tissues

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A253 cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized using 0.1% Triton-X-100 (Sigma-Aldrich) for 10 minutes and blocked with 2% bovine serum albumin (BSA) for 30 minutes, all at room temperature. Then, cells were incubated with a mixture of 10 μg/mL mouse anti-galectin-3 antibody (#ab2785, Abcam, USA) in 2% BSA at 4°C overnight. The next day, cells were incubated with a mixture of 10 μg/mL Alexa Fluor-647 anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., USA) in 2% BSA at room temperature for 1 hour.
Formalin-fixed paraffin embedded sections of murine submandibular glands were deparaffinized, rehydrated and subjected to citric acid microwave antigen retrieval. Slides were blocked with 2% BSA (Sigma-Aldrich) and permeabilized by 0.1% Triton-X-100 (Sigma-Aldrich) for 30 minutes at 25°C. Slides were incubated with mouse anti-galectin-3 (#ab2785, Abcam) and rabbit anti-LAMP1 (#21997-1-AP, Proteintech) antibodies at 4°C overnight, followed by incubation with Alexa Fluor-647 anti-mouse IgG and Alexa Fluor-488 anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc.) at room temperature for 1 hour.
Sections were subsequently counterstained with DAPI (#ab104139, Abcam). Images were acquired using a fluorescent microscope (Nikon, Japan) and analyzed using ImageJ software (public domain, source: National Institutes of Health, USA).
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7

Cell Migration Assay with Scratch Test

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Cells were plated on collagen‐coated 60‐mm culture dishes. Confluent cell sheets were scratched with 200‐μL yellow pipette tips to generate straight‐lined gaps. Each gap was marked with a dot to obtain the same field during image acquisition at 48 and 72 hours after incubation. The scratched area was measured by Image J software (National Institutes of Health).
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8

Viability Assay for Encapsulated oMSCs

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Viability of oMSCs encapsulated into the hydrogels was analysed at day 1 and 7 by double staining with ethidium homodimer-1 and calcein-AM assay according to the manufacturer’s instructions. Briefly, cells were washed with PBS 1X and stained with 500 μL of calcein-AM 2 μM and ethidium homodimer-1 4 μM for 2 h at 37°C. Then samples were rinsed twice with PBS 1X and imaged with a confocal laser scanning microscope (LSM 880, Zeiss, Oberkochen, Germany) at 488 and 543 nm. Cell viability was quantified using Image-J software (National Instruments, Austin, US). Viability data were calculated according to equation (3).
%ofviablecells=Livecells/Totalnumberofcells
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9

Seed Mucilage Secretion Kinetics

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Dry seeds were mounted directly on specific ESEM microscope stubs fitted on a Peltier stage working at 2°C in a Quanta 200 FEG high resolution environmental scanning electron microscope (ESEM; FEI company) under a variable water partial pressure. Images were recorded using the FEI imaging system with an accelerating voltage of 5 kV, at a working distance of 4 mm. In order to control precisely and follow the kinetics of the mucilage secretion process, three successive increase/decreases of the partial pressure in the chamber were applied on the seed. Step by step, the vacuum was adjusted from 3 Torrs where the seeds are considered in extremely dry conditions (30% relative humidity) up to 6 Torrs corresponding to the full seed imbibition in water (100% relative humidity and condensation). Image analysis for slight brightness and contrast improvement was carried out using ImageJ software (W. Rasband, National Institutes of Health).
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10

Western Blot Analysis of Protein Expression

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Total proteins were extracted with RIPA buffer (Thermo Fisher Scientific), supplemented with 1 mm phenylmethylsulfonyl fluoride (Sigma‐Aldrich), and 1× protease inhibitor cocktail (Sigma‐Aldrich). Proteins (50 μg) were resolved via SDS/PAGE and analyzed by western blot using the following antibodies: pRb (IF‐8), p53 (DO1), Myc (SC‐40), ERK2 (SC‐1647), p‐ERK (Tyr‐204) (SC‐7383), AKT1/2/3 (SC‐55523), and p‐AKT (Ser‐473) (SC‐7985‐R) obtained from Santa Cruz Biotechnology (Dallas, TX, USA). CCND1 (AB16663), GAPDH (AB8245), ODC1 (AB66067), P‐eIF4E (Ser‐209) (AB76256), 4EBP1 (AB2606), p‐4EBP1 (Thr‐70) (AB75831), and MNK1 (AB89223) were purchased from Abcam (Cambridge, UK). Antibody‐immobilized membranes were incubated with a corresponding horseradish peroxidase‐conjugated secondary antibody for 2 h. Finally, the immunoreactive proteins were detected using an enhanced chemiluminescent substrate (Millipore, Burlington, MA, USA) and imaged through a gel documentation system (ImageQuant LAS 500; GE Healthcare Life Science, Björkgatan, Sweden). Furthermore, the expression of proteins was quantified by measuring the density of bands using imagej software (National Institutes of Health, Bethesda, MD, USA).
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