欢迎您, 登录 | 注册

首页 | English

X
加载中

This protocol is a more detailed version of previous protocols (Yang et al., 2011; Bolaños-Villegas et al., 2013) developed for the examination of meiotic chromosome spreads. Meiotic chromosome spreads are useful to determine the presence of defects in chromosome pairing and segregation. The protocol also describes how to perform fluorescent in situ hybridization experiments with a centromere probe used to label chromosomes.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

Protocol for the Preparation of Arabidopsis Meiotic Chromosome Spreads and Fluorescent in situ Hybridization
拟南芥减数分裂染色体的分离制备方法和荧光原位杂交技术

植物科学 > 植物细胞生物学 > 细胞成像
作者: Pablo Bolaños-Villegas
Pablo Bolaños-VillegasAffiliation: Academia Sinica, Institute of Plant and Molecular Biology, Taipei, Taiwan
Bio-protocol author page: a1286
Xiaohui Yang
Xiaohui YangAffiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, OH, USA
Bio-protocol author page: a1287
Christopher A. Makaroff
Christopher A. MakaroffAffiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, OH, USA
Bio-protocol author page: a1288
 and Guang-Yuh Jauh
Guang-Yuh JauhAffiliation: Academia Sinica, Institute of Plant and Molecular Biology, Taipei, Taiwan
For correspondence: jauh@gate.sinica.edu.tw
Bio-protocol author page: a511
Vol 4, Iss 8, 4/20/2014, 4952 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1102

[Abstract] This protocol is a more detailed version of previous protocols (Yang et al., 2011; Bolaños-Villegas et al., 2013) developed for the examination of meiotic chromosome spreads. Meiotic chromosome spreads are useful to determine the presence of defects in chromosome pairing and segregation. The protocol also describes how to perform fluorescent in situ hybridization experiments with a centromere probe used to label chromosomes.
Keywords: Meiocyte(性细胞), Cell cycle(细胞周期), Centromere(着丝粒), Reproduction(繁殖), Higher plants(高等植物)

[Abstract]

Materials and Reagents

  1. Inflorescences from young, healthy Arabidopsis thaliana plants
  2. Agar plates, with 1/2x Murashige and Skoog solid medium
  3. Pectolyase (Sigma-Aldrich, catalog number: P3026 )
  4. Cellulase (Sigma-Aldrich, catalog number: C1184 )
  5. β-glucoronidase (Roche Diagnostics, catalog number: 03707598001 )
  6. Sucrose (Affymetrix, catalog number: 21938 )
  7. Ethanol (EMD Millipore, catalog number: 1085430250 )
  8. Chloroform (EMD Millipore, catalog number: 102442 )
  9. Glacial acetic acid (EMD Millipore, catalog number: 100063 )
  10. Citric acid anhydrous (Affymetrix, catalog number: 13729 )
  11. Sodium citrate dihydrate (Affymetrix, catalog number: 13735 )
  12. Formamide (Sigma-Aldrich, catalog number: F9037 )
  13. Saline-sodium citrate (SSC) (Sigma-Aldrich, catalog number: S0902 )
  14. Dextran sulfate (Sigma-Aldrich, catalog number: 42867 )
  15. Salmon sperm (Life Technologies, catalog number: 15632-011 )
  16. VECTASHIELD® mounting medium with DAPI (10 ml) (Vector Laboratories, catalog number: H-1400 )
  17. Fluorescein High-Prime labeling kit (Roche Diagnostics, catalog number: 11585622910 )
  18. pAL1 centromere probe (ABRC, catalog number: CD3-16 )
  19. Taq DNA polymerase with standard Taq buffer (New England Biolabs, catalog number: M0273S )
  20. QIAquick Gel Extraction Kit (kit for purification of DNA from gels) (QIAGEN, catalog number: 28704 )
  21. Gel Pilot 1 Kb Ladder (molecular weight marker for DNA) (QIAGEN, catalog number: 239085 )
  22. Agarose for electrophoresis (Bio-Rad Laboratories, catalog number: BR 161-3100 )
  23. Carnoy’s solution (see Recipes)
  24. 10 mM Sodium citrate buffer (see Recipes)
  25. Enzyme digestion buffer (see Recipes)
  26. FISH buffer (see Recipes)
  27. 10x PBS (see Recipes)
  28. 20x SSC (see Recipes)

Equipment

  1. Hypodermic needles and syringe (Terumo Medical Corporation, model: 26G x ½”, catalog number: NN-2613R )
  2. Tweezers (Dumont)
  3. Poly-Prep® poly-L-lysine coated slides (Sigma-Aldrich, catalog number: P0425-72EA )
  4. Nunc® Lab-Tek® II slides (Thermo Fisher Scientific, catalog number: 154453 )
  5. Coverslips (Matsunami Glass, catalog number: C218181 )
  6. Hybridisation oven OV1 (Biometra, catalog number: 052-090 )
  7. Thermal cycler (Biometra, catalog number: 050-551 )
  8. Dry block (Thermo Fisher Scientific, Reacti-Therm®)
  9. -86 °C refrigerator (Thermo Fisher Scientific, model: Forma® 88000 )
  10. Frigidaire -20 °C refrigerator (Frigidaire, model: FCFS201LFB )
  11. Fluorescence microscope
  12. Coplin staining jars (Thermo Fisher Scientific, catalog number: 107 )
  13. Rubbermaid LunchBlox® food containers
  14. Super Pap Pen’ liquid blocker pen (Electron Microscopy Sciences, catalog number: 71310 ) (optional)

Procedure

  1. Preparation of Arabidopsis meiotic chromosome spreads
    Note: The most important aspect of this experiment is to collect flower buds at the right stage to isolate anthers that contain male meiocytes. Typically flower buds come in racemes, and only 3-4 are usable. Their size cannot exceed 1/4 of the length of the mouth of a Terumo® 26G x ½” hypodermic needle (approx.1 mm). Anthers should be green. In order to prepare a single slide 5-10 flower buds are required.
    1. Prior to anther collection and fixation remove the enzyme digestion solution from the refrigerator and allow it to thaw on ice. In the meantime set a hybridization oven at 37 °C, and prepare a lunch box in which the bottom is covered by moist tissues. On the bottom of the lunch box place a rack for PCR tubes, and place the slides on top to avoid direct contact with the wet tissues. Digestion of the anthers allows free meiocytes to be released onto slides where they can be stained and observed under the microscope.
    2. Racemes are placed first on 1/2x Murashige and Skoog agar plates. Flower buds are excised with tweezers and then placed in a drop of double distilled water (20 μl) in a Lab-Tek® II slide. Lab-Tek® II slides are perfect for this experiment since their coating keeps water drops in place, something regular poly-L-lysine slides cannot do.
    3. To remove water or any other solution, press the mouth of the needle against the slide, and lift the piston of the syringe slowly (the piston can be lifted slightly before placing the syringe against the slide). Choose an area away from the buds. If possible use the tweezers to stretch the droplet away from the buds.
    4. Once all flower buds are placed on water, work can begin. Individual flower buds are moved onto a dry area within the slide. After 10 sec the buds mostly dry out and can be dissected at ease. For dissection two tools are used: a tweezer and two hypodermic needles (26G x ½”). Flower buds are placed horizontally and cut at the base with the needles. This allows for quick removal of the petals. Once petals are removed the anthers can be moved individually into a drop of water with the aid of the needles. It is important to maintain the needles dry to allow easy manipulation of the remaining anthers. Yellow anthers are not meiotic anthers and should not be used at all. Anthers must remain moist at all times.
    5. Once anthers are all placed together, water is removed, leaving only 5 μl. In this way all buds can be further aggregated with the needle. Then add Carnoy’s fixative 20 μl for 20 min, and replenish every 5 min to avoid evaporation (as an option the Super Pap Pen’ liquid blocker pen can be used to better contain the Carnoy’s fixative). Carnoy´s fixative contains no formaldehyde, gluteraldehyde or chloroform, therefore it has little toxicity and allows subsequent ease of manipulation. After 20 min have the fixative removed but leave at least 5 μl. Aggregate the anthers. Then quickly add water, and have it quickly removed (but never totally, leave 5 μl). Then change water two more times (5 min each).
    6. Add citrate buffer (20 μl). This buffer is used to prepare anthers for enzymatic digestion.
    7. After removal of citrate buffer add 20-40 μl of enzyme solution to the slide, place a Lab-Tek® II plastic cap on top of the slide (each slide comes with a cap), place the slides inside the lunch box, close it well, and have it placed inside the hybridization oven. Allow digestion of the buds for 2 h at 37 °C. Once digestion is finished, remove the slides, and remove most of the solution (leave 5 μl). Then quickly add 20 μl of citrate buffer, and have it quickly removed (never totally, leave 5 μl), then change the citrate buffer two more times (5 min each) and leave only 10 μl on the slide. Remove the citrate buffer slowly and carefully. If citrate is removed too quickly most anthers will be removed as well and the slides may contain few or no meiotic spreads.
    8. At this point use the needle, or the tweezers to squeeze the anthers. This allows the release of free meiocytes onto the slide. Do it slowly, for about 10 min. It is important to squeeze slowly and carefully; otherwise the anthers will adhere to each other. At this point the solution will turn turbid, which is normal. Add buffer if needed.
    9. Add 20 μl of 60% acetic acid, and mix slowly. The solution will turn transparent. Do not allow to evaporate. Acetic acid causes meiocytes to swell, thereby improving the quality of the final chromosome spreads. Transfer 5 μl of the resulting cells suspension to four different poly-L-lysine slides. Place a glass coverslip on top, but before placing it carefully misalign the coverslip by 45 degrees to either side to allow protrusion of one corner. Fold a piece of paper tissue and place a slide inside, then press firmly from above with your thumb. It is very important to keep your thumb in place to prevent displacement of the coverslip. Application of pressure allows bursting of the cells and removal of cytoplasm, while the use of paper tissue prevents the slide from breaking while applying pressure from above.
    10. Move the slides to a -80 °C refrigerator, and leave them there for at least 10 min. Then take them out quickly and remove the coverslip with the help of a razor. Allow the slides to air dry at room temperature.
    11. Once the slides are dry add 5-30 μl of DAPI solution (vectashield with mounting medium for instance) to one of the 4 slides and cover with a new coverslip. Observe one slide under a fluorescence microscope to confirm whether the slide contains good meiotic chromosome spreads, if so the remaining slides can be used for FISH experiments. Meiotic stages can be identified using Figure 1 as a template (previously unpublished). You may seal the edges of the coverslip with regular nail polisher for short-term storage in the dark. Vectashield contains a mounting medium that allows for short term storage.


      Figure 1. Stages of male meiosis in Arabidopsis as visualized by DAPI staining. In the wild type five bivalents are always observed during metaphase-I. Scale bar: A-D, 20 μm, E, F, H, 5 μm, G, I-L, 10 μm

  2. FISH (fluorescence in situ hybridization)
    1. Prepare 70% formamide in 2x SSC (700 μl formamide, 100 μl 2x SSC and 100 μl distilled water). Add 50 μl to each slide, place a coverslip on top and place the slides on a hot dry block set at 80 °C. After 30 sec you may either a) dip the slide on water inside a Coplin jar to dislodge the coverslip, or b) apply 20 μl of water to lift the coverslip. Then place the slides in a Coplin jar filled with 200 ml of 70%, 90% and 100% ethanol, for 5 min each. Please notice that all ethanol solutions must be cooled in advance at -20 °C. After the bath series is finished allow the slides to air dry at room temperature.
    2. In the meantime prepare the pAL1 centromere probe (for this step you require a fully labeled probe). Concentration of probe: 20-30 ng/μl. Dissolve 1.5 μl in 13.5 μl of FISH buffer (for one slide), for a final FISH probe concentration of approximately 2-3 ng/μl (see Notes). Incubate the probe at 80 °C for 5-10 min to denature DNA. Spin shortly afterwards with a microcentrifuge (at approximately 10,000 rpm), at room temperature. Then quickly place the FISH probe mix on ice for at least 5 min.
    3. Add 15 μl of FISH probe onto each slide. Cover with a coverslip and incubate the slides on the lunch box overnight at 37 °C. The bottom of the box should contain wet paper tissues to maintain a high humidity and avoid dessication of the FISH probe.
    4. Next morning dip the slides on water to remove the coverslip, and then place the slides on a Coplin jar. Wash with 200 ml 2x SSC for 10 min, four times.
    5. You can check the condition of the slides at this point by taking one and have it stained with DAPI.
    6. Wash the slides on 200 ml hot 2x SSC (45 °C) for 10 min, and after that in 200 ml 1x PBS for 5 min. Washing with PBS is not considered essential and can be skipped.
    7. Add DAPI (5 μl) while the sample is wet. Cover with a coverslip and observe. Results can be compared with Figure 2 (previously unpublished).
    8. If for some reason cells are washed away, you can wash the cells on 2x SSC for 15 min once and then directly stain with DAPI.


      Figure 2. Arabidopsis male meiotic spread (zygotene stage) hybridized to the pAL1 centromeric 180 bp repeat (yellow) and counterstained with DAPI (red, pseudocolor). The number of centromere signals (4) roughly corresponds to the actual number of bivalents (5). Scale bar: 10 μm

Notes

  1. How to prepare a centromere probe
    In order to prepare the centromere probe, PCR must be performed with specific primers (M13 forward and reverse). These two primers are required for the amplification of the Arabidopsis 180 bp centromeric repeat using the centromere-containing plasmid, pAL1 as a template. The primers are as follows:
    M13-Forward: 5’-GTAAAACGACGGCCAG-3’
    M13-Reverse: 5’-CAGGAAACAGCTATGAC-3’
    Ordinarily, the pAL1plasmid is diluted1/100x for PCR and the resulting product is labeled for 8 h with the Fluorescein High-Prime labeling kit. Please read the manufacturer´s instructions carefully.

Recipes

  1. Carnoy’s solution (v/v)
    95% ethanol, chloroform and glacial acetic acid (6:3:1)
  2. 1 L 10 mM Sodium citrate buffer
    1.045 g citric acid anhydrous (MW = 192.12)
    1.479 sodium citrate dihydrate (MW = 294.1)
  3. Enzyme digestion buffer
    0.3% pectolyase Y23, 1.5 mg (0.0015 g)
    0.3% cellulase, 1.5 mg (0.0015 g)
    1.4 % β-glucoronidase, 7.5 mg (0.0075 g)
    6% sucrose, 30 mg (0.03 g)
    First dissolve enzymes in 10 mM sodium citrate (100 μl), and then continue to add sodium citrate up to 500 μl. This solution can be stored for up to one month at -20 °C.
  4. FISH buffer (500 μl)
    10% formamide (add 50 μl 100% formamide)
    2x SSC (add 50 μl 20x SSC)
    10% dextran sulfate (add 100 μl 50% dextran sulfate, which is prepared by simply dissolving 1 g of dextran sulfate powder in 2 ml of distilled water, then heat to 80 °C to improve mixing)
    Salmon sperm blocking DNA (add 75 μl of 25x sperm, final concentration: 250 μg/μl)
    In order to prepare 25x salmon sperm DNA (25 μg/μl), dissolve 250 mg sperm powder into 1 ml of distilled water or 1x PBS
    Keep at -20 °C
    0.1% SDS (add 5 μl of 10% SDS)
    Distilled water, 220 μl
  5. 10x phosphate buffered saline (PBS)
    Prepare NaCl, 80 g; KCl, 2 g; Na2HPO4.7H2O, 14.4 g; KH2PO4, 2.4 g; ddH2O up to 1,000 ml
    Adjust pH to 7.2-7.6
    Autoclave
  6. 20x SSC
    Prepare NaCl, 175.3 g (3 M), and sodium citrate dihydrate 88.2 g (300 mM), then dissolved in 800 ml of water
    Adjust pH to 7.0
    Take volume to 1,000 ml
    Autoclave

Acknowledgments

This work was supported by research grants from Academia Sinica (Taiwan), the National Science and Technology Program for Agricultural Biotechnology (NSTP/AB, 098S0030055-AA), Taiwan, and the National Science Council (NSF; 99-2321-B-001-036-MY3), Taiwan, to G.-Y. Jauh; an NSF grant (MCB0718191) to C. A. Makaroff, and a study-abroad contract from the University of Costa Rica to P. Bolaños-Villegas.

References

  1. Bolaños-Villegas, P., Yang, X., Wang, H. J., Juan, C. T., Chuang, M. H., Makaroff, C. A. and Jauh, G. Y. (2013). Arabidopsis CHROMOSOME TRANSMISSION FIDELITY 7 (AtCTF7/ECO1) is required for DNA repair, mitosis and meiosis. Plant J 75(6): 927-9.
  2. Yang, X., Boateng, K. A., Yuan, L., Wu, S., Baskin, T. I. and Makaroff, C. A. (2011). The radially swollen 4 separase mutation of Arabidopsis thaliana blocks chromosome disjunction and disrupts the radial microtubule system in meiocytes. PLoS One 6(4): e19459.

材料和试剂

  1. 来自年轻健康的拟南芥植物的花序
  2. 琼脂平板,用1/2x Murashige和Skoog固体培养基
  3. 果胶酶(Sigma-Aldrich,目录号:P3026)
  4. 纤维素酶(Sigma-Aldrich,目录号:C1184)
  5. β-葡萄糖醛酸酶(Roche Diagnostics,目录号:03707598001)
  6. 蔗糖(Affymetrix,目录号:21938)
  7. 乙醇(EMD Millipore,目录号:1085430250)
  8. 氯仿(EMD Millipore,目录号:102442)
  9. 冰醋酸(EMD Millipore,目录号:100063)
  10. 无水柠檬酸(Affymetrix,目录号:13729)
  11. 柠檬酸钠二水合物(Affymetrix,目录号:13735)
  12. 甲酰胺(Sigma-Aldrich,目录号:F9037)
  13. 盐水 - 柠檬酸钠(SSC)(Sigma-Aldrich,目录号:S0902)
  14. 硫酸葡聚糖(Sigma-Aldrich,目录号:42867)
  15. 鲑鱼精子(Life Technologies,目录号:15632-011)
  16. (Vector Laboratories,目录号:H-1400)的VECTASHIELD 安装介质中。
  17. 荧光素High-Prime标记试剂盒(Roche Diagnostics,目录号:11585622910)
  18. pAL1着丝粒探针(ABRC,目录号:CD3-16)
  19. 使用标准Taq 缓冲液(New England Biolabs,目录号:M0273S)进行Taq DNA聚合酶
  20. QIAquick凝胶提取试剂盒(用于从凝胶纯化DNA的试剂盒)(QIAGEN,目录号:28704)
  21. Gel Pilot 1 Kb Ladder(DNA的分子量标记)(QIAGEN,目录号:239085)
  22. 琼脂糖电泳(Bio-Rad Laboratories,目录号:BR161-3100)
  23. Carnoy的解决方案(见配方)
  24. 10 mM柠檬酸钠缓冲液(参见配方)
  25. 酶消化缓冲液(参见配方)
  26. FISH缓冲区(参见配方)
  27. 10x PBS(请参阅配方)
  28. 20x SSC(请参阅配方)

设备

  1. 皮下注射针和注射器(Terumo Medical Corporation,型号:26G×1/2",目录号:NN-2613R)
  2. 镊子(杜蒙)
  3. 聚-L-赖氨酸包被的载玻片(Sigma-Aldrich,目录号:P0425-72EA)
  4. Nunc Lab-Tek II载玻片(Thermo Fisher Scientific,目录号:154453)
  5. 盖玻片(Matsunami Glass,目录号:C218181)
  6. 杂交炉OV1(Biometra,目录号:052-090)
  7. 热循环仪(Biometra,目录号:050-551)
  8. 干块(Thermo Fisher Scientific,Reacti-Therm )
  9. -86℃冰箱(Thermo Fisher Scientific,型号:Forma 88000)
  10. Frigidaire -20℃冰箱(Frigidaire,型号:FCFS201LFB)
  11. 荧光显微镜
  12. Coplin染色瓶(Thermo Fisher Scientific,目录号:107)
  13. Rubbermaid LunchBlox ®食品容器
  14. Super Pap Pen液体阻滞笔(Electron Microscopy Sciences,目录号:71310)(可选)

程序

  1. 拟南芥减数分裂染色体扩散的制备
    注意:这个实验的最重要的方面是收集花蕾在正确的阶段,以隔离包含男性meiocytes的花药。通常花芽进入消旋,只有3-4是可用的。它们的尺寸不能超过Terumo 26G x½"皮下注射针(约1mm)的嘴长度的1/4。花药应为绿色。为了准备一张幻灯片,需要5-10个花蕾。
    1. 在花药收集和固定之前,从冰箱中取出酶消化溶液,并使其在冰上解冻。同时将杂交炉设置在37℃,并制备其中底部被潮湿组织覆盖的饭盒。在午餐盒的底部放置用于PCR管的架,并将载玻片放置在顶部,以避免与湿组织直接接触。花药的消化允许将自由的细胞释放到载玻片上,在显微镜下可以染色和观察。
    2. 将总状花序首先置于1/2x Murashige和Skoog琼脂平板上。用镊子切除花芽,然后置于Lab-Tek II载玻片的一滴双蒸水(20μl)中。 Lab- Tek ® II载玻片对于这个实验是完美的,因为它们的涂层使水滴保持在原位,而正常的聚-L-赖氨酸载玻片不能做到。
    3. 要清除水或任何其他溶液,将针头压在滑块上,缓慢提起注射器的活塞(在将注射器靠在滑块上之前,活塞可以稍微提起)。选择一个远离芽的区域。如果可能,使用镊子将液滴从芽上拉开
    4. 一旦所有的花蕾都放在水上,工作就可以开始了。将个体花蕾移动到载玻片内的干燥区域上。 10秒后,芽主要变干,可以轻松解剖。为了解剖,使用两个工具:镊子和两个皮下注射针(26G×1/2")。将花芽水平放置,并用针在基部切割。这允许快速移除花瓣。一旦瓣被移除,花药可以借助于针而单独地移动到一滴水中。重要的是保持针干燥以允许容易地操纵剩余的花药。黄色花药不是减数性花药,不应该使用。花药必须始终保持湿润。
    5. 一旦花药都放在一起,水被去除,只留下5微升。以这种方式,所有芽可以进一步与针聚集。然后添加Carnoy的固定剂20微升20分钟,每5分钟补充,以避免蒸发(作为一个选项超级Pap笔的液体阻滞笔可以用于更好地包含Carnoy的固定剂)。 Carnoy的固定剂不含甲醛,戊二醛或氯仿,因此它几乎没有毒性,并允许随后易于操作。 20分钟后,固定剂被移除,但留下至少5微升。聚集花药。然后迅速加水,并迅速删除(但从不完全,离开5微升)。然后再换水两次(每次5分钟)。
    6. 加入柠檬酸盐缓冲液(20μl)。该缓冲液用于制备用于酶消化的花药。
    7. 去除柠檬酸盐缓冲液后,向载玻片添加20-40μl酶溶液,将一个Lab-Tek ® II塑料盖放在载玻片的顶部(每个载玻片带有盖),将载玻片在饭盒内,关好它,并将其放置在杂交炉内。允许芽在37℃下消化2小时。一旦消化完成,删除幻灯片,并删除大部分的溶液(留5μl)。然后快速加入20微升柠檬酸盐缓冲液,并迅速删除(从不完全,离开5微升),然后更改柠檬酸盐缓冲液两次(每次5分钟),并留下只有10微升在幻灯片上。缓慢小心地除去柠檬酸盐缓冲液。如果柠檬酸盐被去除太快,大多数花药也将被去除,并且载玻片可能含有很少或没有减数分裂的扩散。
    8. 此时使用针,或镊子挤压花药。这允许将游离的毛细胞释放到载玻片上。慢慢地,大约10分钟。重要的是慢慢地仔细挤压;否则花药会相互粘附。此时溶液会变浑浊,这是正常的。如果需要,添加缓冲区。
    9. 加入20μl的60%乙酸,慢慢混合。解决方案将变得透明。不允许蒸发。乙酸引起毛细胞肿胀,从而提高最终染色体扩散的质量。转移5微升的所得细胞悬浮液到四个不同的聚-L-赖氨酸幻灯片。将玻璃盖玻片放置在顶部,但在放置之前仔细错开盖玻片45度到任一侧,以允许一个角的突出。折叠一张纸巾并将幻灯片放在里面,然后用拇指从上面用力按压。保持你的拇指到位以防止盖玻片的位移是非常重要的。施加压力允许细胞破裂和去除细胞质,而使用纸巾防止载玻片在从上方施加压力时断裂。
    10. 将载玻片移动到-80°C冰箱,并留在那里至少10分钟。然后迅速拿出来,并用剃刀帮助除去盖玻片。让载玻片在室温下风干。
    11. 一旦幻灯片干燥添加5-30微升的DAPI解决方案(vectashield与封装介质例如)到4个幻灯片之一,并盖上一个新的盖玻片。观察一个幻灯片在荧光显微镜下,以确认幻灯片是否包含良好的减数分裂染色体扩散,如果这样,剩下的幻灯片可以用于FISH实验。可以使用图1作为模板鉴定减数分裂期(先前未公开)。您可以用常规指甲抛光器密封盖玻片的边缘,以便在黑暗中短期储存。 Vectashield包含允许短期存储的封装介质

      图1.通过DAPI染色显现的 拟南芥 中的男性减数分裂阶段。总是在中期I期间观察到。比例尺:A-D,20μm,E,F,H,5μm,G,I-L,10μm

  2. FISH(荧光原位杂交)
    1. 在2×SSC(700μl甲酰胺,100μl2×SSC和100μl蒸馏水)中制备70%甲酰胺。添加50微升到每个幻灯片,盖上盖玻片,将幻灯片放置在设置在80℃的热干燥块。 30秒后,您可以a)将幻灯片放在Coplin罐内的水上,以移除盖玻片,或b)应用20μl的水提起盖玻片。然后将载玻片置于装有200ml 70%,90%和100%乙醇的Coplin罐中,每个5分钟。请注意,所有乙醇溶液必须提前在-20°C冷却。浴系列完成后,让载玻片在室温下风干。
    2. 同时准备pAL1着丝粒探针(对于这一步,你需要一个完全标记的探针)。探针浓度:20-30 ng /μl。溶解1.5μl在13.5μlFISH缓冲液(一个幻灯片),最终FISH探针浓度约2-3 ng /μl(见注释)。孵育探针在80℃下5-10分钟使DNA变性。之后立即用微量离心机(约10,000rpm)在室温下旋转。然后快速将FISH探针混合物置于冰上至少5分钟
    3. 在每个载玻片上加入15μlFISH探针。盖上盖玻片,并在37°C下孵育午餐盒上的载玻片过夜。盒子底部应包含湿纸巾,以保持高湿度,避免FISH探针的干燥
    4. 第二天早晨将载玻片浸在水上以除去盖玻片,然后将载玻片放在Coplin瓶子上。用200ml 2x SSC洗涤10分钟,四次。
    5. 您可以通过拍摄一张幻灯片来检查幻灯片的状态,并用DAPI对其进行染色
    6. 在200 ml热2x SSC(45°C)上洗涤载玻片10分钟,然后在200 ml 1 x PBS中5分钟。用PBS清洗不是必需的,可以跳过
    7. 当样品湿润时,加入DAPI(5μl)。盖上盖玻片并观察。结果可以与图2(先前未公开)比较。
    8. 如果由于某些原因细胞被洗掉,你可以在2x SSC上洗涤细胞15分钟一次,然后用DAPI直接染色。


      拟南芥 拟南芥 男性减数分裂(zygotene阶段)与pAL1着丝粒180bp重复(黄色)杂交并用DAPI (红色,假色)。着丝粒信号(4)的数量大致对应于二价物的实际数量(5)。比例尺:10μm

笔记

  1. 如何准备着丝粒探针
    为了制备着丝粒探针,必须用特异性引物(正向和反向的M13)进行PCR。 使用含有着丝粒的质粒pAL1作为模板,需要这两个引物用于扩增拟南芥180bp着丝粒重复。 引物如下:
    M13-正向:5'-GTAAAACGACGGCCAG-3'
    M13-反向:5'-CAGGAAACAGCTATGAC-3'
    通常,将pAL1质粒稀释1/100x进行PCR,所得产物用荧光素High-Prime标记试剂盒标记8小时。 请仔细阅读制造商的说明。

食谱

  1. Carnoy溶液(v/v)
    95%乙醇,氯仿和冰醋酸(6:3:1)洗脱
  2. 1升10mM柠檬酸钠缓冲液 1.045g无水柠檬酸(MW = 192.12) 1.479柠檬酸钠二水合物(MW = 294.1)
  3. 酶消化缓冲液
    0.3%果胶酶Y23,1.5mg(0.0015g) 0.3%纤维素酶,1.5mg(0.0015g) 1.4%β-葡萄糖醛酸酶,7.5mg(0.0075g) 6%蔗糖,30mg(0.03g) 首先溶解酶在10mM柠檬酸钠(100μl)中,然后继续加入柠檬酸钠至500μl。该溶液可在-20°C下储存一个月。
  4. FISH缓冲液(500μl)
    10%甲酰胺(加入50μl100%甲酰胺) 2×SSC(加50μl20×SSC) 10%硫酸葡聚糖(加入100μl50%硫酸葡聚糖,通过简单地将1g硫酸葡聚糖粉末溶解在2ml蒸馏水中制备,然后加热至80℃以改善混合) 鲑鱼精子阻断DNA(加入75μl25x精子,最终浓度:250μg/μl)
    为了制备25x鲑鱼精子DNA(25μg/μl),将250mg精子粉末溶解于1ml蒸馏水或1x PBS中
    保持在-20°C
    0.1%SDS(加5μl10%SDS) 蒸馏水,220μl
  5. 10x磷酸盐缓冲盐水(PBS)
    制备NaCl,80g; KCl,2g; Na 2 HPO 4 sub。7 7H 2 O,14.4g;实施例1的化合物。 KH 2 PO 4 4,2.4g; ddH <2> O高达1000ml
    将pH调节至7.2-7.6
    高压灭菌器
  6. 20x SSC
    制备175.3g(3M)的NaCl和88.2g(300mM)柠檬酸钠二水合物,然后溶解在800ml水中
    将pH调节至7.0
    取体积到1000 ml
    高压灭菌器

致谢

这项工作得到了中国台湾研究院,台湾农业生物技术国家科技计划(NSTP/AB,098S0030055-AA)和国家科学委员会(NSF; 99-2321-B-001 -036-MY3),台湾,到G.-Y.高雅一个NSF资助(MCB0718191)给C.A.Makaroff,以及一个从哥斯达黎加大学到P.Bolaños-Villegas的留学合同。

参考文献

  1. Bolaños-Villegas,P.,Yang,X.,Wang,H.J.,Juan,C.T.,Chuang,M.H.,Makaroff,C.A。和Jauh,G.Y。 DNA需要拟南芥色素发酵蛋白7(AtCTF7/ECO1)修复,有丝分裂和减数分裂。植物J 75(6):927-9。
  2. Yang,X.,Boateng,K.A.,Yuan,L.,Wu,S.,Baskin,T.I.和Makaroff,C.A。(2011)。 径向肿胀4 分离酶突变的拟南芥(Arabidopsis thaliana)阻断染色体断裂并破坏微生物中的径向微管系统。 6(4):e19459。
English
中文翻译

免责声明

为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。

X


How to cite this protocol: Bolaños-Villegas, P., Yang, X., Makaroff, C. A. and Jauh, G. (2014). Protocol for the Preparation of Arabidopsis Meiotic Chromosome Spreads and Fluorescent in situ Hybridization. Bio-protocol 4(8): e1102. DOI: 10.21769/BioProtoc.1102; Full Text



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


登陆 | 注册
引用格式
分享
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook