Anthers samples were collected from male fertile plants (wild type) and sterile plants (ms1d) containing meiotic microspores (meiosis I, dyad and tetrad) and uninucleate microspores. Developmental stages were determined by acetocarmine staining or cytological examination. Callose wall staining was performed by squashing anthers in a drop of aniline blue solution (0.1% aniline blue in 0.1 M phosphate buffer pH 7.0) [45 (link)]. Both bright-field and fluorescence microscopy were performed using a Nikon ECLIPSE NiE optical microscope.
Eclipse nie optical microscope
Manufactured by Nikon
The ECLIPSE NiE optical microscope is a high-performance laboratory instrument designed for a variety of microscopy applications. It features advanced optics and illumination systems to provide clear, high-quality images. The ECLIPSE NiE is capable of diverse imaging techniques, including brightfield, darkfield, and phase contrast microscopy.
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4 protocols using eclipse nie optical microscope
1
Meiotic Microspore Developmental Stages
2
Quantifying Ovary Mesocarp Cell Characteristics
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Ovary samples collected at different flowering times were fixed overnight in FAA (50% ethanol, 5% acetic acid, 4% formaldehyde), and then stored in 70% ethanol at 4 °C until processing. Ovary samples were dehydrated through an ethanol series and the samples were subsequently embedded in paraffin as described elsewhere. The 10 μm thin sections (cross or longitudinal) of ovary were prepared using a Leica RM2265 rotary microtome at Adelaide Microscopy Facility. The sections were stained by 0.05% toluidine blue solution (Soukup, 2014 ) and mounted with DPX Mountant (Fluka Analytical, Switzerland). Images of ovary sections were taken by using a Nikon ECLIPSE NiE optical microscope. The number of ovary mesocarp cells was counted from the longitudinal (ventral to dorsal) section images. A line was drawn at the maximum ovary depth position in the image, and the number of cells under the line was counted. For ovary mesocarp cell size, the perimeters of 10 randomly selected mesocarp cells at the top front position in the longitudinal sections were measured (in total, 80–110 cells per sample).
3
Polyethylene Glycol-Mediated Protoplast Transfection
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Polyethylene glycol (PEG)-mediated transfections were mostly carried out as described by Bai et al. (2014) . Firstly, 200 μl of PEG–Ca2+ solution [40% (w/v) PEG 4000, 0.4 M mannitol, 0.1 M CaCl2] were pre-loaded in pipette tips. Secondly, 100 μl of protoplast solution (~105 cells) were added to 20 μg of each plasmid DNA in a 2.0 ml tube. The pre-loaded PEG–Ca2+ solution was immediately added to the protoplast–DNA mixture, mixed gently, and incubated for 15 min at room temperature in the dark. The transfection process was stopped by adding 840 μl of W5 and centrifuged at 600 g for 2 min. Cells were resuspended in 500 μlof W5 and transferred to multi-well plates previously coated with 5% (v/v) foetal bovine serum (FBS). Protoplasts were cultured at 28 °C for 40–48 h in the dark.
To test transfection efficiency, protoplasts were transfected with pUbi-YFP-rbcS in six independent transfections. At 40–48 h after transfection, the protoplasts were visualized under UV and bright light using a Nikon Eclipse Ni-E optical microscope equipped with a DS-Ri1 colour cooled digital camera.
To test transfection efficiency, protoplasts were transfected with pUbi-YFP-rbcS in six independent transfections. At 40–48 h after transfection, the protoplasts were visualized under UV and bright light using a Nikon Eclipse Ni-E optical microscope equipped with a DS-Ri1 colour cooled digital camera.
4
Light and Electron Microscopy of Carpels
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For light microscopy, wild-type, mov2, and mov1 carpels were fixed in FAA solution (50% ethanol, 5% glacial acetic acid, 10% formaldehyde, one drop of Tween-20) overnight, dehydrated in a 70–100% ethanol series, and embedded in LR white resin. Samples were sectioned using a Leica Rotary Microtome RM2265 at 1.5 μm. Slides were stained with 0.1% toluidine blue in 0.1% sodium tetraborate for 2 min and rinsed three times with water, dried, and mounted with DPX. After 72 h, slides were imaged with a Nikon Eclipse Ni-E optical microscope equipped with a DS-Ri1 colour cooled digital camera. Image analysis and processing were carried out with the NIS-Elements AR software.
For SEM, inflorescences were manually dissected and fixed overnight in 4% paraformaldehyde, 1.25% glutaraldehyde in phosphate-buffered saline (PBS), 4% sucrose, pH 7.2. Before processing, samples were washed three times in PBS and fixed in 2% OsO4 in PBS for 1 h. Samples were then dehydrated in a 50–100% ethanol series and dried with a critical point dryer. Dried samples were arranged on carbon tabs stuck to 12 mm aluminium stubs and coated with platinum. Samples were observed using a Hitachi FlexSem 1000 scanning electron microscope.
For SEM, inflorescences were manually dissected and fixed overnight in 4% paraformaldehyde, 1.25% glutaraldehyde in phosphate-buffered saline (PBS), 4% sucrose, pH 7.2. Before processing, samples were washed three times in PBS and fixed in 2% OsO4 in PBS for 1 h. Samples were then dehydrated in a 50–100% ethanol series and dried with a critical point dryer. Dried samples were arranged on carbon tabs stuck to 12 mm aluminium stubs and coated with platinum. Samples were observed using a Hitachi FlexSem 1000 scanning electron microscope.
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