Callose deposition in the leaf was measured according to the method from a previous study [64 (link)]. Leaves from different treated sets were fixed and distained in acetic acid/ethanol (1:3) until the sample was transparent. After 12 h, the saturated distaining solution was replaced. The leaf sample was then incubated in 150 mM K2HPO4 for 30 min for washing. After washing, the samples were rinsed with 150 mM K2HPO4 and 0.01% aniline blue (staining solution), then incubated for 2 h in dark at room temperature. Finally, the sample was mounted in 50% glycerol and observed under fluorescence microscope using DAPI filter. The optimal excitation wavelength was set at 370 nm and the emission wavelength ranges between 490 and 510 nm in the Floid Cell Imaging Station microscope (Life Technologies).
Floid cell imaging station microscope
Manufactured by Thermo Fisher Scientific
The Floid Cell Imaging Station is a microscope designed for high-quality imaging of cells. It provides advanced optical capabilities to capture detailed images of cellular structures and processes.
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8 protocols using floid cell imaging station microscope
1
Quantifying Leaf Callose Deposition
2
Pathogen Penetration and Colonization Assay
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Fresh, healthy 30 days old bean leaves were collected and were surface-sterilized using 0.01% HgCl2 solution for 2 min. After two successive washes with sterile, distilled water, the leaves were placed in a sterile Petri dish and kept moist with wet blotting paper. The leaves were then inoculated with 10 μl of conidial suspension (1 × 106 conidia ml−1). Simultaneously, a control set was prepared by inoculating the leaf with sterile, distilled water in a similar way. The Petriplates were incubated for 5 days at 25 °C in the dark. The pathogen was re-isolated from diseased leaf and maintained in PDA.
To study the pathogen penetration and colonization process, inoculated leaf pieces approximately 6 × 6 mm were cut and immersed in a clearing solution of absolute ethanol/glacial acetic acid (1:2) overnight to remove chlorophyll [36 (link)]. The cleared leaf was mounted on glass microscopic slides in clear glycerin and observed under white and green filters of a Floid Cell Imaging Station microscope (Life Technologies).
To study the pathogen penetration and colonization process, inoculated leaf pieces approximately 6 × 6 mm were cut and immersed in a clearing solution of absolute ethanol/glacial acetic acid (1:2) overnight to remove chlorophyll [36 (link)]. The cleared leaf was mounted on glass microscopic slides in clear glycerin and observed under white and green filters of a Floid Cell Imaging Station microscope (Life Technologies).
3
Lignin Auto-Fluorescence Microscopy
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Lignin production was recorded by the auto-fluorescence microscopy, as described in a previous study [65 (link)]. According to the author, lignin has a wide array of fluorescence emission and is excited by UV rays. Lignified cell walls of xylem tissue show auto-fluorescence. Thin hand sections of leaf petiole of different treated leaves were made with a fine blade and mounted in 50% glycerol. Auto-fluorescence of lignin was visualized with the help of a Floid Cell Imaging Station microscope (Life Technologies) under the excitation wavelength 330 nm to 380 nm and emission wavelength between 500 and 530 nm.
4
Visualizing Real-time Nitric Oxide Production
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Real time NO production was visualized using a DAF-2DA membrane permeable fluorochrome dye [59 ]. Thin sections of roots were taken in loading buffer and 50 µL of 10 mM KCl and 50 µL of 10 mM TrisHCl (pH 7.2) with final concentration of DAF-2DA 10 mM were added and incubated in the dark for 20 min. Fluorescence was observed and high resolution images were taken using Floid Cell Imaging station microscope by Life technologies. Green fluorescence indicates the production of NO.
5
Nitric Oxide Measurement in Plant Tissues
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Production of NO was measured by hemoglobin assay according to the method from a previous study [37 (link)]. Leaf tissues of control and treated sets were incubated in a reaction mixture containing 10 mM L-arginine and 10 mM hemoglobin in a total volume of 5 mL of 0.1 M phosphate buffer (pH 7.4). Production of NO was measured spectrophotometrically at 401 nm and NO levels were calculated using an extinction coefficient of 38,600 M−1cm−1. After 2 h of incubation, NO content in the reaction mixture was measured as nmol of NO produced g−1 tissue h−1.
Real-time NO production was observed by using membrane permeable flurochrome 4-5 diaminofluorescein diacetate (DAF-2DA) dye [58 ]. The lower epidermis of the leaf was peeled off and thin sections were prepared from the petiole and placed in a brown bottle containing 1 mL of loading buffer containing 10 mM KCl and 10 mM Tris HCl (pH 7.2) with DAF-2DA at a final concentration of 10 mM for 20 min in dark. Fluorescence was observed with a Floid Cell Imaging Station microscope (Life Technologies) at excitation wavelength of 480 nm and emission wavelength of 500–600 nm. Green fluorescence indicates the production of NO.
Real-time NO production was observed by using membrane permeable flurochrome 4-5 diaminofluorescein diacetate (DAF-2DA) dye [58 ]. The lower epidermis of the leaf was peeled off and thin sections were prepared from the petiole and placed in a brown bottle containing 1 mL of loading buffer containing 10 mM KCl and 10 mM Tris HCl (pH 7.2) with DAF-2DA at a final concentration of 10 mM for 20 min in dark. Fluorescence was observed with a Floid Cell Imaging Station microscope (Life Technologies) at excitation wavelength of 480 nm and emission wavelength of 500–600 nm. Green fluorescence indicates the production of NO.
6
Quantifying ROS Levels in Plant Roots
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ROS generation was monitored according to the method of Gupta et al. [60 (link)]. To measure ROS, thin transverse sections of treated roots were immersed in the 1 mL of detection buffer DB (2.5 mM HEPES, pH 7.4) containing 10 µM DCF-2DA fluorescent dye (Invitrogen, Carlsbad, CA, USA), and it was kept for 10 min in dark incubation. The high-resolution images were checked using Floid Cell Imaging station microscope by Life technologies.
7
Peptide-Mediated Transfection of HCT-116 Cells
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Herein, 1 mL of complete medium containing 2 × 105 HCT-116 cells was grown in six-well plates. The sterile glass coverslips were kept in each well of six-well tissue culture plates before the cell seeding. The Alexa 488 bDNA (0.04 nmol) was mixed with 4 nmol of peptides in 1 mL of serum-free DMEM medium and kept at room temperature for 30 min to form a peptide-bDNA complex. Then complexes were introduced to cells and incubated at 37°C in 5% CO2 for 4 h. After incubation, medium was taken out, and cells were fixed with 4% paraformaldehyde followed by PBS wash. In 1:5,000 dilution, DAPI was added for nucleus staining. With the aid of mounting material, the coverslips were fixed. The FLoid Cell Imaging Station microscope from Life Technologies was then used to study the cells.
8
In vivo detection of root H2O2
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In vivo detection of H2O2 of root samples was carried out using DAB by following the method of Thordal-Christensen et al. [61 (link)]. After one week of germination the roots were excised and immersed in a solution containing 1 mg/mL diaminobenzidine (DAB) solution (pH 3.8) and incubated for 8 h in the dark. After that sectioning of roots were performed and the section is immersed in 3:1 (V/V) ethanol and glacial acetic acid mixture for bleaching. After that it was cleaned with water and for 24 h the sections were dipped in lactoglycerol (1:1:1, lactic acid: glycerol:water V/V). High-resolution images were taken using Floid Cell Imaging station microscope by Life technologies.
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