Immunohistochemistry images obtained by Olympus FV1200 were next analyzed by Image-J (NIH, MD USA: https://imagej.nih.gov/ij/ ). Signal intensities (AU/pixel) of YAP, YAPdeltaC, phospho-LATS1 and phospho-PLK1 in each neuron (NeuN-positive or MAP2-positive cell) were quantified by free-hand-surrounding the shape of neuron with Image-J. From human immunohistochemistry images 4 visual fields were randomly selected, and 100 neurons in total were analyzed. Background signals were collected from 8 areas that did not include cells, and the signal intensity of each neuron was subtracted with the mean value of the background signals. The mean value of the signal intensities of 100 neurons after subtraction of the background signals was used as the representative value for a patient or a control, and statistical analysis was performed between 3 patients and 3 controls.
Imagej
Manufactured by Olympus
Sourced in Japan, United States, Germany, Italy
ImageJ is an open-source image processing software designed for scientific use. It provides a platform for the visualization, analysis, and processing of digital images and microscopy data. The software's core function is to enable researchers to perform a variety of image-related tasks, such as image segmentation, measurement, and quantification, without any promotional or biased interpretation.
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Lab products found in correlation
Fluorescence microscope, by Olympus (5 mentions)
Microsuite, by Olympus (3 mentions)
Fv1000 confocal microscope, by Olympus (3 mentions)
Fluorescence microscopy, by Olympus (3 mentions)
Cellsens software, by Olympus (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Confocal microscope, by Olympus (2 mentions)
Fluoview analysis software, by Olympus (2 mentions)
Bz x710, by Olympus (2 mentions)
Cx43 microscope, by Olympus (2 mentions)
Ottix plus, by Diapath (2 mentions)
Ottix sharper solvents, by Diapath (2 mentions)
Lysotracker red, by Thermo Fisher Scientific (2 mentions)
Hm525 nx, by Thermo Fisher Scientific (2 mentions)
Inverted microscope, by Olympus (2 mentions)
Fluoview fv1000 confocal laser scanning microscope, by Olympus (2 mentions)
Fv1000, by Olympus (2 mentions)
Ab32064, by Abcam (1 mentions)
Axio imager widefield fluorescence microscope, by Zeiss (1 mentions)
93 protocols using imagej
1
Quantitative Analysis of Neuronal Protein Levels
2
Quantifying Focal Ischemic Injury in Rodents
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MAP2-stained sections were analyzed to quantify grey matter injury [32–34 (link)]. For quantification of infarct size, every 50th section throughout the brain was stained for MAP2. Images were captured using an Olympus light microscope with 1.25× magnification and analyzed using ImageJ (version 1.51j8, ImageJ.nih.gov/ij">http:/ImageJ.nih.gov/ij , National Institutes of Health, U.S.A.). Infarct size was calculated by subtracting the ipsilateral MAP2-stained area from the contralateral and expressing the difference as percentage of the stained contralateral area [32 (link)]. Quantifications were made for the whole hemisphere and for cortex specifically. All quantifications were performed in a blinded manner.
Iba-1 stained sections were analyzed by phenotype scoring, using a modified version of a previously described scoring method [35 (link),36 (link)]. Briefly, affected brain regions (striatum, upper and lower cortex, cerebral peduncle, globus pallidus and substantia nigra) were given a score 0-4, based on microglial phenotype and accumulation (Supplementary Figure S1). Each region was scored using a light microscope (Olympus) with three 20× magnified pictures at each level. A mean score for each region was calculated at each level, and then a mean for the region throughout the different levels in which the region was present.
Iba-1 stained sections were analyzed by phenotype scoring, using a modified version of a previously described scoring method [35 (link),36 (link)]. Briefly, affected brain regions (striatum, upper and lower cortex, cerebral peduncle, globus pallidus and substantia nigra) were given a score 0-4, based on microglial phenotype and accumulation (Supplementary Figure S1). Each region was scored using a light microscope (Olympus) with three 20× magnified pictures at each level. A mean score for each region was calculated at each level, and then a mean for the region throughout the different levels in which the region was present.
3
Measuring L4 worm body size
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For the body size measurement, the L1 worms were exposed to the different exposure concentrations of the target samples and then allowed to reach adulthood. L4 worm images were captured using a dissecting microscope (Olympus, SZX10, Waltham MA, USA) at a 1.5X magnification, and the sizes were measured using the Java-based open source imaging software, ImageJ (http://ImageJ.nih.gov/ij/) (Wisconsin, USA). Three biological replicates were performed, and a total of 150 worms for each experimental group were measured.
4
Histological Assessment of Hepatic Steatosis
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After 3 weeks of treatment with DOCA-salt, the mice were euthanized and the livers were excised, fixed in 4% paraformaldehyde, and embedded in paraffin. The paraffin embedded tissues were sectioned at 4 μm and stained with hematoxylin and eosin (HE) by standard methods. Hepatic steatosis was blindly assessed on 4 random fragments from different areas of each liver and was staged on a scale of 0 to 4, according to the percentage of hepatocytes containing cytoplasmic vacuoles as follows: 0 (<5%), 1 (5–20%), 2 (20%–30%), 3 (30–60%), and 4 (≥60%). Frozen samples were also stained with Oil-Red-O at the optimal cutting temperature. Slides were analyzed by microscopy using Image J and Microsuite (Olympus Soft Imaging Solutions GmbH, Munster, Germany).
5
Root Hair Growth Stimulation Assay
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For the root hair growth stimulation assay by pathogenic bacteria, the experiments were performed as described in (Pečenková et al., 2017a (link)). Briefly, inoculation was performed with P. syringae pv. maculicola ES4326 (Psm) as follows: cultures from freshly inoculated plates (Luria–Bertani medium with 25 mg/l of rifampicin and streptomycin) were used to prepare liquid overnight culture (40 ml; with incubation at 28°C on an orbital shaker at 130 rpm). Bacteria were centrifuged at 1,500 g for 10 min and the pellet was resuspended in sterile distilled water (dH2O) and diluted to an OD600 of 0.3 (108 CFU/ml). Approximately 10 μl droplets were applied to cover the root tips within the elongation and differentiation zone. As a mock control, dH2O was applied in the same manner and, 48 h later, the root hair growth was inspected. Root hair lengths were analyzed using AnalySIS (Soft Imaging System GmbH, Germany) or ImageJ (Schindelin et al., 2015 (link)) software. The numerical data obtained (sample sizes for each of the lines are presented in Supplementary Table 2
6
Histopathological Analysis of Liver Samples
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Sections of formalin-fixed livers were stained with hematoxylin-eosin and Sirius Red. Frozen samples were also stained with Oil-Red-O that was made at the optimal cutting temperature. Immunohistochemistry staining for cleaved PARP (Abcam, cat. # ab32064), alpha fetoprotein (AFP) (Abcam cat. # ab46799) or sonic hedgehog (shh) (Santa Cruz cat. # sc-9024) was performed in formalin-fixed, paraffin-embedded liver sections according to the manufacturer’s specifications. Slides were analyzed using microscopy using ImageJ and Microsuite (Olympus Soft Imaging Solutions GmbH, Munster, Germany). This histopathology was independently analyzed by a veterinary pathologist (JKP) in a blinded manner (see “Author contributions”).
7
Histological Assessment of Liver Pathology
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The livers were fixed in 10% buffered formalin, processed, and embedded in paraffin for hematoxylin-eosin (H&E) staining. Frozen samples prepared at the optimal cutting temperature were also stained with Oil Red O. The microscope slides were analyzed using ImageJ and Microsuite (Olympus Soft Imaging Solutions GmbH, Munster, Germany).
Histological scoring of hepatic steatosis, lobular inflammation, ballooning, and fibrosis was performed by pathologists who were blinded to the group assignments.
Histological scoring of hepatic steatosis, lobular inflammation, ballooning, and fibrosis was performed by pathologists who were blinded to the group assignments.
8
Colony Formation Assay of ACHN and OS-RC-2 Cells
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ACHN and OS-RC-2 cells (0.1 × 103 cells per well) were seeded in a six-well plate and cultured for 10 days after treatment. Colonies were then fixed with 4% paraformaldehyde for 15 min and stained for 10 min with 0.5% crystal violet. Then, the number of colonies was counted using ImageJ, and images were taken under an Olympus microscope (Tokyo, Japan).
9
Evaluating Cardiac Development Toxicity
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Tg (cmlc: EGFP) zebrafish, which specifically expresses the enhanced green fluorescent protein (EGFP) in myocardial cells, was used to examine the changes in the distance between sinus venosus (SV) and bulbus arteriosus (BA) to indicate the cardiac development toxicity [25 (link)]. In this study, the adverse effects of 2,6-DHNPs on cardiomyogenesis were performed. Briefly, 10 larvae of Tg (cmlc: EGFP) zebrafish in each treatment were randomly selected and anesthetized (0.168 mg/mL MS-222) for 1 min. Subsequently, the distance from sinus venosus to bulubs arteriosus (μm) was measured at 72 hpf using a fluorescent microscope (BX43, Olympus, Japan) and quantified by Image J (Bethesda, MD).
10
Immunohistochemistry Staining Protocol
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To perform IHC staining, the tissues were cut into 4 μm sections. The slides were deparaffinized and incubated with citrate buffer. After being treated with H2O2 to inhibit endogenous peroxidase, the sections were blocked with diluted BSA. The antibodies used for IHC staining are shown in the supplementary Table 4 . Finally, IHC images were taken by a microscope (Olympus Co., Tokyo, Japan), and the positive fields were quantified with ImageJ.
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