After being embedded in paraffin, specimens of the distal colon were sectioned at 5 µm, mounted on glass slides and stained with hematoxylin and eosin for the histopathology analysis. Digital images at 40× magnification per section were captured with a Zeiss Axiphot light microscope (Carl Zeiss MicroImaging LLC, Thornwood, NY) and a Nikon D1X digital camera (Tokyo, Japan). Five fields per section were examined to determine the morphological lesions and changes in the colon mucosa. The degree of IBD was measured by the modified scoring system of Iba et al. [23] (link). Inflammatory colitis was scored from 0 to 3 for lesions based on loss of epithelium, length of crypts, and infiltration of leukocytes (Table 3 ). The total histological score ranged from 0 to 9, which represented the summed scores of loss of epithelium, length of crypts and infiltration of leukocytes, with a higher score indicating more-severe disease.
D1 x digital camera
Manufactured by Nikon
Sourced in Japan
The D1-x is a digital camera designed for professional use. It features a 2.7-megapixel CCD sensor and supports JPEG and RAW image formats. The camera is capable of capturing images with a resolution of 2,000 x 1,312 pixels. It has a built-in autofocus system and supports burst shooting at a rate of up to 4.5 frames per second.
Automatically generated - may contain errors
Lab products found in correlation
Axiphot light microscope, by Zeiss (1 mentions)
Axiophot light microscope, by Zeiss (1 mentions)
Gel mount aqueous mounting medium, by Merck Group (1 mentions)
Fluorescein isothiocyanate (fitc), by Santa Cruz Biotechnology (1 mentions)
Goat anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
8 protocols using d1 x digital camera
1
Histological Assessment of Inflammatory Colitis
2
Root Growth Inhibition Assays in Arabidopsis
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Arabidopsis thaliana Col-0 was the genetic background of wild-type and mutant lines used in this study. Knock-out lines for coi1-1 (Xie et al., 1998 (link)), coi1-30 (Yang et al., 2012 ), coi1-2 (Xu et al., 2002 (link)), jar1-1 (Staswick and Tiryaki, 2004 (link)), myc2/jin1-2 (Lorenzo et al., 2004 (link)), and jai3-1 (Chini et al., 2007 (link)) have been described previously. Seeds were surface-sterilized by the chlorine gas method (in the presence of bleach and HCl for 3 h) and stratified for 2–3 d at 4 °C in the dark. All of the seedlings were grown under a 16-h light/8-h dark cycle at 21 °C (photosynthetic photon flux density approximately 100 µmol m–2 s–1). For root-growth inhibition assays, 10 to 30 seeds of each line were germinated for 10 d, with or without the presence of 50 µM jasmonic acid, 50 µM LasA, or 0.5 µM COR (Reveglia et al., 2018 (link)). Images were taken with a Nikon D1-x digital camera and root length was estimated using the ImageJ software (https://imagej.nih.gov/ij/ ). Data were analysed by one-way ANOVA/Tukey HSD post hoc tests. Four independent biological replicates (10–30 seedlings each) were measured for each sample with similar results. Experiments were repeated four times with similar results.
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Arabidopsis thaliana Col-0 was the genetic background of wild-type and mutant lines used in this study. Knock-out lines for coi1-1 (Xie et al., 1998 (link)), coi1-30 (Yang et al., 2012 ), coi1-2 (Xu et al., 2002 (link)), jar1-1 (Staswick and Tiryaki, 2004 (link)), myc2/jin1-2 (Lorenzo et al., 2004 (link)), and jai3-1 (Chini et al., 2007 (link)) have been described previously. Seeds were surface-sterilized by the chlorine gas method (in the presence of bleach and HCl for 3 h) and stratified for 2–3 d at 4 °C in the dark. All of the seedlings were grown under a 16-h light/8-h dark cycle at 21 °C (photosynthetic photon flux density approximately 100 µmol m–2 s–1). For root-growth inhibition assays, 10 to 30 seeds of each line were germinated for 10 d, with or without the presence of 50 µM jasmonic acid, 50 µM LasA, or 0.5 µM COR (Reveglia et al., 2018 (link)). Images were taken with a Nikon D1-x digital camera and root length was estimated using the ImageJ software (
3
Yeast Two-Hybrid Protein Interaction Assay
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All constructs were previously reported by Chini et al. (2007) (link). pGAD (prey) and pGBK (bait) plasmids were co-transformed in Saccharomyces cerevisiae AH109 cells using standard heat-shock protocols (Chini et al., 2007 (link)). Successfully transformed colonies were selected 3 d after transformation on yeast synthetic drop-out medium lacking Leu and Trp (–2 medium). Yeast colonies were then grown in selective –2 liquid medium for ~5 h and cell density adjusted to 3 × 107 cells ml–1 (OD600=1). Cell suspensions (5 µl) were plated on yeast synthetic drop-out medium lacking Ade, His, Leu, and Trp (–4 medium) to evaluate protein interactions. Where indicated, the medium was supplemented with 20 µM coronatine and/or 100 µM LasA. Plates were incubated (2–6 d; 28 °C) and images were acquired with a Nikon D1-x digital camera.
4
Marchantia polymorpha growth experiments
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M. polymorpha accession Takaragaike-1 (Tak-1; male) was used in this study as the wild-type (WT). Mpcoi1-2 [18] was used as a mutant in this study. M. polymorpha gemmae were grown on half Gamborg's B5 1% agar medium under continuous light (50-60 mmol m -2 s -1 ) at 21 C, or 30 C where specified. For the 21 C and 30 C growth experiments, the hormone was incorporated into the media throughout the growth period. The quantitative data for this experiment were obtained by measuring the area of the plant with ImageJ software, and growth percentage taken as a ratio of hormone treated versus untreated plants, in each respective temperature. Plant pictures were taken with a NIKON D1-x digital camera. Klebsormidium nitens NIES-2285 was obtained from the Microbial Culture Collection (MCC) stock center at the National Institute for Environmental Studies of Japan (NIES). K. nitens was maintained on half Gamborg's B5 medium 1% agar. Every 3-4 weeks, the algae fully-grown on the surface of the plate were scraped and ground with mortar and pestle at room temperature in approximately 10 mL liquid half Gamborg's B5. The suspension was spread onto several half Gamborg's B5 plates. K. nitens was grown under long-day conditions (16-h light/8-h dark) and 21 C.
5
Silencing Suppression Assay for Viral Proteins
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Silencing suppression assays were performed as described previously (Johansen and Carrington, 2001 ) by infiltration of Agrobacterium tumefaciens cultures carrying the constructs pK7WG2:GFP (expressing the reporter protein GFP), the pK7WG2:HC-Pro-PVY (expressing the known suppressor HC-Pro, as a positive control) and pK7WG2 with inserts corresponding to the complete coding regions of the CP and MP. Negative controls were agroinfiltrated with the pK7WG2:GFP construct only. GFP fluorescence was monitored under UV and leaves were photographed from 3 days to 8 days post-infiltration using a Nikon D1X digital camera.
6
Histopathological Analysis of Kidney Injury
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The kidneys were harvested, fixed with 4% paraformaldehyde, and embedded in paraffin. Specimens were sliced into 5 μm-thick sections and stained with hematoxylin-eosin (H&E) for the histopathological analysis. Digital images at 200x magnification per section were captured with a Zeiss Axiophot light microscope (Carl Zeiss MicroImaging LLC, Thornwood, NY, USA) and a Nikon D1X digital camera (Tokyo, Japan). Five fields per section were examined to determine the morphological changes and lesions. The degree of kidney injury was measured by a semiquantitative scoring system according to Kuruş et al. [18 (link)]. The total histological score ranged from 0 to 8, which represented the summed scores of interstitial fibrosis and tubular injury in the renal cortex (Table 1 ).
7
Immunofluorescence Staining of Cell-Cell Junctions
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Following DMSO or 30 µM 15d-PGJ2 treatment at 37°C, the CGTH W-2 cells were washed with PBS, then fixed for 5 min with 10% formalin in PBS and permeabilized for 10 min with 0.1% Triton X-100 in PBS at room temperature. Following the PBS washes (3 times for 5 min each), nonspecific binding was blocked with a 30 min incubation at room temperature with 5% non-fat milk in PBS. The cells were incubated overnight at 4°C with monoclonal mouse antibodies against p120-ctn (dilution, 1:100; cat. no., 610133) or β-ctn (dilution, 1:100; cat. no., 610153; BD Biosciences, Franklin Lakes, NJ, USA), or vinculin (dilution, 1:100; cat. no., V4505; Sigma-Aldrich; Merck Millipore) washed with PBS and incubated for 1 h at 37°C with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (dilution, 1:50; cat. no., 715–095-151; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). For F-actin labeling, the cells were incubated with FITC-phalloidin (dilution, 1:100; cat. no., P5282; Sigma-Aldrich; Merck Millipore). Finally, the cells were washed with PBS, mounted with Gel Mount™ Aqueous mounting medium (Sigma-Aldrich; Merck Millipore) and detected using a fluorescent microscope (DM2500; Leica Microsystems GmbH, Wetzlar, Germany) and images were captured using a Nikon D1X digital camera (Nikon Corporation, Tokyo, Japan).
8
Colon Mucosa T Cell Subtyping
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Double-staining combinations CD3-CD4 and CD3-CD8 were performed on 5 μm paraffin-embedded colon sections. After antigen retrieval, sections were incubated with an antibody against CD3ε (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C and amplified with a rabbit anti-goat immunoglobulin G (IgG) secondary antibody conjugated with FITC (Santa Cruz Biotechnology). For colocalization, sections were then costained overnight at 4°C with secondary antibodies against CD4 (Abcam, Cambridge, UK) or CD8 (Novus Biologicals, Littleton, CO, USA) and amplified with the respective appropriate secondary antibodies: goat anti-mouse IgG or goat anti-rabbit IgG conjugated with rhodamine (Santa Cruz Biotechnology). Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma, St. Louis, MO, USA) for 10 min at room temperature. Digital images at 400x magnification per section were acquired using appropriate filters of a Zeiss Axiophot fluorescence microscope (Carl Zeiss MicroImaging LLC, Thornwood, NY, USA) fitted with a Nikon D1X digital camera (Tokyo, Japan). Cells containing both FITC and rhodamine labels appeared yellow. These images were then overlaid with DAPI-staining images to determine the infiltration of T lymphocyte subpopulations in the colon mucosa.
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