研究目的
To provide detailed practical steps required to perform immunostaining of barley meiocytes (pollen mother cells) for confocal or structured illumination microscopy, aiming to better understand meiosis and recombination in barley and other grasses.
研究成果
The protocol enables the detailed study of meiosis and recombination in barley through immunostaining and advanced microscopy techniques, providing insights into the control of crossover formation in large genome species.
研究不足
The use of immunocytochemistry in large genome crops like barley has been limited and remains novel. The protocol's effectiveness may vary with different barley cultivars and growing conditions.
1：Experimental Design and Method Selection:
The protocol involves immunostaining of barley meiocytes for confocal or structured illumination microscopy to study meiosis and recombination.
2：Sample Selection and Data Sources:
Barley cv. Bowman plants were grown under controlled conditions, and meiocytes were collected from developing anthers.
3：List of Experimental Equipment and Materials:
Includes orbital shaker, incubator, dark wet chamber, fine forceps, glass rod, glass Coplin staining jar, embryo dish, cavity slide, centrifuge tubes, cryostat, microscope slides, coverslips, and various solutions like aceto-carmine, PBS, SSC, fixative, blocking solution, anti-fading agent, primary and secondary antibodies.
4：Experimental Procedures and Operational Workflow:
Detailed steps for meiocyte preparation, immunostaining protocol, and section preparation are provided, including fixation, washing, antibody incubation, and microscopy.
5：Data Analysis Methods:
The prepared samples are analyzed using confocal or structured illumination microscopy to observe meiotic events.
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Alexa Fluor secondary antibodies
A10042, A21206, A11077, A21208
Thermo Fisher Scientific
Used for detecting primary antibodies in immunostaining.
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Cryostat
CM1100
Leica
Used for sectioning samples.
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Confocal microscope
LSM 710
Carl Zeiss
Used for analyzing chromosome behavior during cell division.
ZEISS LSM 990 Spectral Multiplex
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Orbital shaker
Used for mixing samples during preparation.
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4°C incubator
Used for incubating samples at controlled temperatures.
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Dark wet chamber
Used for incubating slides during immunostaining.
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Fine forceps
Used for dissecting anthers.
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Glass rod
Used for tapping out fixed anthers to release meiocytes.
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Glass Coplin staining jar
Used for staining samples.
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Embryo Dish
30 mm Diameter
Used for holding samples during preparation.
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Cavity Slide
Single Depression
Used for holding samples during preparation.
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Centrifuge tubes
1.5 mL, 2 mL, and 50 mL
Used for sample storage and preparation.
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Microscope slides
Polysine or Superfrost plus
Used for mounting samples for microscopy.
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Glass coverslip
22 × 50 mm, thickness No 1.5
Used for covering samples on slides.
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Glass coverslip
18 × 18 (or 22 × 22), thickness No 1 or 1.5
Used for covering samples on slides.
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Dark Dropping bottle
Used for storing aceto-carmine solution.
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Aluminum slide tray
20 slides capacity
Brunel Microscopes Ltd.
Used for storing slides.
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Razor blade
single edge
Used for cutting samples.
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Tissue paper
Used for removing excess staining solution.
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Nail varnish
Used for sealing coverslips on slides.
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