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Analysis of Chromosome Condensation/Decondensation During Mitosis by EdU Incorporation in Nigella damascena L. Seedling Roots
通过EdU掺入分析大马士革黑种草幼苗根中有丝分裂过程中染色体的凝集/解凝集   

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参见作者原研究论文

本实验方案简略版
BMC Plant Biology
Jan 2017

Abstract

To investigate the chromosome dynamics during mitosis, it is convenient to mark the discrete chromosome foci and then analyze their spatial rearrangements during prophase condensation and telophase decondensation. To label the chromosome regions in plant chromosomes, we incorporated the synthetic nucleotide, 5-ethynyl-2’-deoxyuridine (EdU), which can be detected by click-chemistry, into chromatin during replication. Here, we described a protocol of a method based on the application of semi-thin sections of Nigella damascena L. roots embedded in LR White acrylic resin. The thickness of semi-thin (100-250 nm) sections is significantly lower than that of optical sections even if a confocal microscope was used. This approach may also be suitable for work with any tissue fragments or large cells (oocytes, cells with polytene chromosomes, etc.).

Keywords: Plant (植株), Chromosome (染色体), Replication (复制), EdU (EdU), Click-chemistry (点击化学)

Background

Most data concerning chromosome organization have been acquired from studies of a small number of model organisms, the majority of which are mammals. In plants with large genomes, the chromosomes are significantly larger than the animal chromosomes that have been studied to date. To investigate the chromosome dynamics during mitosis, it is necessary to mark the discrete chromosome foci and then analyze their spatial rearrangements during prophase condensation and telophase decondensation. To label the chromosome regions, we incorporated the synthetic nucleotide, 5-ethynyl-2’-deoxyuridine (EdU) (Kuznetsova et al., 2017). Detection of EdU is based on a click-reaction, which is a copper catalyzed reaction between an azide and an alkyne. The EdU contains the alkyne which can react with the azide-containing detection reagent.

The most suitable distribution of labeled regions (i.e., separated labeled dots) was seen in cells which incorporated EdU during late S-phase. The brief pulse labeled all S-phase cells, and the initial appearance of EdU-labeled mitotic figures thus denoted the time needed for cells labeled in late S-phase to traverse into mitosis.

Root apical meristem does not allow for the acquisition of high-resolution images because of the out-of-focus fluorescence. Here, we described a protocol of a method based on the application of semi-thin (100-250 nm) sections of roots embedded in LR White acrylic resin. LR White is a polyhydroxy-aromatic acrylic resin with low toxicity and ultra-low viscosity. The polymerized resin is hydrophilic (sections freely permeable to aqueous solutions). The thickness of semi-thin sections is significantly lower than that of optical sections even if a confocal microscope was used. This approach may also be suitable for work with any tissue fragments or large cells (e.g., cells with polytene chromosomes).

Materials and Reagents

  1. 30 mm and 90 mm Petri dishes (Greiner Bio One International)
  2. Filter paper
  3. Single edge blades (Ted Pella, catalog number: 121-3 )
  4. 1.5 microtubes (SSIbio, catalog number: 1260-00 )
  5. Snap-fit Gelatin Capsules, Size 2 (Ted Pella, catalog number: 130-19 )
  6. Cover slips, No. 1 (Fisher Scientific, catalog number: 12-548A )
  7. Microscope slides (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: AA00000112E00MNT10 )
  8. Parafilm
  9. Cotton wool
  10. Foil
  11. Safety gloves
  12. Nigella damascena L. seeds
  13. Click-iT EdU Alexa 555 Imaging Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: C10338 )
  14. Thymidine (Sigma-Aldrich, catalog number: T9250 )
  15. Phosphate buffered saline (PBS), pH 7.2 (10x) (Thermo Fisher Scientific, GibcoTM, catalog number: 70013016 )
  16. Ethanol
  17. 4’,6-Diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 62248 )
  18. Tris base
  19. Concentrated HCl
  20. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
  21. LR White embedding kit (Sigma-Aldrich, catalog number: 62662 )
    Note: This product has been discontinued.
  22. Formvar 1595E (Serva, catalog number: 21740 )
  23. 1,2-Dichloroethane anhydrous (Sigma-Aldrich, catalog number: 284505 )
  24. Mowiol 4-88 (Sigma-Aldrich, catalog number: 81381 )
  25. Glycerol (MP Biomedicals, catalog number: 04800687 )
  26. 1,4-Diazabicyclo-[2.2.2]-octane (Sigma-Aldrich, catalog number: D2522 )
    Note: This product has been discontinued.
  27. Deionized H2O
  28. 1 M Tris-HCl (pH 8.5) (see Recipe 1)
  29. Paraformaldehyde (see Recipe 2)
  30. LR White acrylic resin (see Recipe 3)
  31. Formvar coated cover slips (see Recipe 4)
  32. Mowion mounting medium (see Recipe 5)

Equipment

  1. Perfect Loop (Ted Pella, catalog number: 13064 )
  2. Orbital Shaker OS-20 (Biosan, model: OS-20 , catalog number: BS-010108-AAG)
  3. Laboratory incubator TC1/20 (SKTB, model: TC-1/20 , catalog number: 1003)
  4. Chemical fume hood
  5. Ultratome LKB III
  6. Fluorescent microscope Axiovision 200M (Carl Zeiss, model: Axiovision 200M ) equipped with the ORCAII-ERG2 camera (Hamamatsu).
    Note: For deconvolution, AxioVision 3.1 software (Carl Zeiss) was used.
  7. pH meter
  8. Magnetic stirrer with hot plate MSH-300 (Biosan, model: MSH-300, catalog number: BS-010302-OAA )
  9. Glass or diamond knife
  10. Micro-centrifuge MiniSpin (Eppendorf, model: MiniSpin® plus , catalog number: 5452000018)

Procedure

  1. Germinate the seeds of Nigella damascena L. in 90 mm Petri dishes covered with damp filter paper at 25 °С in the dark. It takes about a week for roots to grow into 10 mm.
  2. For chromatin labeling, use 10 mm long roots. Incubate the germinated seeds in distilled water with 50 μM EdU (EdU is a part of Click-iT EdU Alexa 555 Imaging Kit, prepare the stock solution according to manufacturer’s recommendations) for 30 min. Incubate in 90 mm Petri with 10 ml of EdU solution on an orbital shaker (50 rpm) mounted inside an incubator at 25 °С.
  3. Incubate seeds in 200 µM thymidine for 30 min, and then grow in distilled water during different time period that allowed to label different chromosome regions. In case of Nigella damascena L., 3.5 h incubation is sufficient to find the first labeled mitosis (Kuznetsova et al., 2017). Incubate in 90 mm Petri with 10 ml of thymidine solution or distilled water on an orbital shaker (50 rpm) mounted inside an incubator at 25 °С.
  4. Excise root tips that are 1.0 mm long from 10 mm long roots using a single edge blade, transfer to 1.5 ml microtubes and fix in 2% paraformaldehyde in 0.5x PBS for 1.5 h at room temperature (for additional fixation protocol see Note 1).
    Note: Use a chemical fume hood while handing formaldehyde-containing chemicals.
  5. After fixation, wash the root tips in 0.5x PBS, dehydrate and infiltrate in LR White acrylic resin using the following steps:
    1. Wash roots with 0.5x PBS 3 times for 5 min each.
    2. Wash roots with 50% ethanol for 30 min.
    3. Dehydrate specimens in 70% ethanol 2 times for 30 min each (it is possible to store specimens in 70% ethanol at 4 °С).
    4. Infiltrate with LR White and 70% ethanol (2:1) mixture (1 ml) for 1 h.
      For LR white/ethanol mixture preparation, slowly add one part of 70% ethanol (drop by drop) to two parts of LR White, and rotate the tube gently (otherwise the mixture will become milky).
    5. Incubate in LR White (1 ml) for 1 h.
    6. Incubate in LR White (1 ml) overnight at 4 °С.
    7. Incubate in LR White (1 ml) for 1 h.
    Note: Incubate without rocking and at room temperature, unless otherwise specified.
  6. It is important to limit the contact of oxygen with the resin while polymerization occurs. The most convenient way of achieving this is to use gelatin capsules. Place several (3-4) roots in the bottom of the capsule and fill up with LR White to the brim and slide the other half of the capsule on (Figure 1).


    Figure 1. The transfer of roots from microtubes to gelatin capsules for polymerization. For removal of roots from microtubes and transfer to capsules, it is convenient to use straight tip tweezers and do not discard LR White form the microtubes. Fill up the gelatin capsules with LR White to the brim.

  7. Polymerize at 55 °С for 24 h.
  8. Prepare semi-thin sections (100-250 nm) using an Ultratome using either glass knife or diamond knife for sectioning of histology specimens.
  9. Mount semi-thin sections on Formvar-coated cover slips (see Recipes) using a Perfect Loop.
  10. Dry cover slips with semi-thin sections at 37 °С for 2-3 h for better attachment of section.
  11. Detect EdU using a Click-iT EdU Alexa 555 Imaging Kit according to manufacturer’s protocol (see Note 2). The staining is carried out in the humid chamber in the dark. The humid chamber is a 90 mm non-vented Petri dish, whose bottom is covered with Parafilm. A piece of moist cotton wool is placed near the dish wall. Drops of a dye solution are placed on the Parafilm, cover slips are placed on these drops (sections downwards) (Figure 2). The dishes are sealed with the Parafilm.


    Figure 2. Humid chamber

  12. Stain DNA with 1 μg/ml DAPI solution in 30 mm Petri dishes for 10 min, and after washing in PBS for 5 min, mount sections in Mowiol with the anti-bleaching agent, 1,4-diazabicyclo-[2.2.2]-octane (DABCO).
  13. Image stacks may be acquired using either wild-field fluorescent microscopy with 3D-deconvolution (Figure 3).


    Figure 3. Chromosome labeling with EdU. Metaphase cell of Nigella damascena L. whose chromosomes included EdU during late S-phase. Wild-field fluorescent microscopy with 3D-deconvolution. Scale bar = 5 μm.

Notes

  1. It is possible to use glutaraldehyde for fixation (2.5% glutaraldehyde in 50 mM Sörensen buffer). But after such fixation, the pale autofluorescence is clearly visible. We cannot recommend this fixation for conventional light microscopy, but it is possible to use it for high-resolution microscopy and correlative light and electron microscopy.
  2. There are a lot of fluorophore azides from different manufactures (Molecular Probes, Lumiprobe, Sigma-Aldrich, etc.). The selection of fluorochrome for the staining depends on the microscopic system which is used for image acquisition. Our microscope has filter sets optimized for the detection of Alexa 488 and Alexa 555 (or different fluorochromes with similar spectral characteristics). We prefer to use Alexa 555 because of substantially brighter staining which we obtained with this fluorochrome.

Recipes

  1. 1 M Tris-HCl (pH 8.5)
    1. Dissolve Tris base in water (Tris base 121.14 g, deionized water ~800 ml)
    2. Using a pH meter, titrate the solution with concentrated HCl until the correct pH is reached
    3. Add deionized water to 1,000 ml
    4. Store at 4 °C
  2. Paraformaldehyde
    1. Mix 2.0 g of paraformaldehyde with 100 ml of 0.5x PBS in a glass that was tightly closed with foil
    2. Heat the solution on a magnetic stirrer with a hot plate until it becomes transparent (Figure 4). Do not let the solution boil. We prefer to heat with the maximal temperature with the permanent observation
    3. Then the solution will be transparent, it is necessary to remove the glass from the heater. The diluted formaldehyde may be aliquoted, frozen and stored at -20 °C. We never stored the aliquots for more than 1 month. It is important to thaw out each aliquot only one time
    Note: Use a chemical fume hood while handing formaldehyde-containing chemicals.


    Figure 4. Paraformaldehyde dissolution using magnetic stirrer with hot plate

  3. LR White acrylic resin
    1. Add the benzoyl peroxide catalyst (1.998 g) to the LR white acrylic resin (100 ml) at room temperature (both reagents are the parts of LR White embedding kit)
    2. The catalyst will take a full 24 h at room temperature to dissolve completely. During this time, occasionally shaking the activated resin mixture is recommended. Once mixed with catalyst and fully dissolved, the resin must be stored at 2-8 °C to maintain its shelf life but not longer than 9 months
    Note: Work under the chemical fume hood and in safety gloves. If you have direct contact with skin or eyes, wash the affected areas with plenty of water and soap.
  4. Formvar coated cover slips
    3 mg of Formvar is diluted in 10 ml of 1,2-dichloroethane. This solution can be stored in airtight glass in the dark. Cleaned cover slips are dipped in a solution of Formvar and left to dry vertically. Formvar coated cover slips should be prepared before the use
  5. Mowion mounting medium
    1. Add 1.2 g of Mowiol 4-88 to 3 g of glycerol, and then add 3 ml of deionized H2O and leave for 2 h at room temperature
    2. Add 6 ml of 0.2 M Tris-HCl (pH 8.5) and heat to 50 °C with occasional mixing till the Mowiol will be dissolved. Add 1,4-diazabicyclo-[2,2,2]-octane (DABCO) to final concentration 2.5% to reduce fading
    3. Centrifuge the mounting medium at maximal speed in micro-centrifuge for 15 min, and transfer supernatant to the clean microtubes
    4. Aliquot in airtight microtubes can be stored at -20 °C

Acknowledgments

The work was supported by the Russian Science Foundation (project 14-15-00199). The protocol was adapted from previous work (Kuznetsova et al., 2017). The authors declare no conflicts of interest or competing interests that may impact the design and implementation of this protocol.

References

  1. Kuznetsova, M. A., Chaban, I. A. and Sheval, E. V. (2017). Visualization of chromosome condensation in plants with large chromosomes. BMC Plant Biol 17(1): 153.

简介

为了研究有丝分裂期间的染色体动力学,可以方便地标记离散的染色体焦点,然后分析它们在前期缩合和末期解聚期间的空间重排。 为了在植物染色体上标记染色体区域,我们将可以通过点击化学检测的合成核苷酸5-乙炔基-2'-脱氧尿苷(EdU)掺入染色质中。 在这里,我们描述了一种方法的协议,该方法基于嵌入在LR白色丙烯酸树脂中的
【背景】关于染色体组织的大多数数据是从少数模式生物的研究中获得的,其中大部分是哺乳动物。在具有大基因组的植物中,染色体显着大于迄今为止研究的动物染色体。为了研究有丝分裂期间的染色体动力学,有必要标记离散的染色体焦点,然后分析它们在前期凝结和末期解聚过程中的空间重排。为了标记染色体区域,我们引入了合成的核苷酸5-乙炔基-2'-脱氧尿苷(EdU)(Kuznetsova等人,2017)。 EdU的检测基于点击反应,其是叠氮化物和炔烃之间的铜催化反应。 EdU含有可与含叠氮化物的检测试剂反应的炔。

在S期后期并入EdU的细胞中可看到标记区域(即,分离的标记的点)的最合适的分布。短脉冲标记了所有S期细胞,并且EdU标记的有丝分裂图的初始外观因此表示在S期晚期标记的细胞穿过有丝分裂所需的时间。

根尖分生组织由于离焦荧光不允许获取高分辨率图像。在这里,我们描述了一个方法的基础上应用半薄(100-250纳米)部分嵌入LR白色丙烯酸树脂的根。 LR White是一种低毒,超低粘度的多羟基芳香族丙烯酸树脂。聚合树脂是亲水性的(水溶液可自由渗透的部分)。即使使用共焦显微镜,半薄切片的厚度也明显低于光切片的厚度。这种方法也适用于任何组织碎片或大细胞(例如,具有多线染色体的细胞)。

关键字:植株, 染色体, 复制, EdU, 点击化学

材料和试剂

  1. 30毫米和90毫米培养皿(格瑞纳生物一国际)
  2. 滤纸
  3. 单刃刀片(Ted Pella,目录号:121-3)
  4. 1.5 microtubes(SSIbio,目录号:1260-00)
  5. 卡扣明胶胶囊,大小2(泰德佩拉,目录号:130-19)
  6. 封面简介,1号(Fisher Scientific,目录号:12-548A)
  7. 显微镜载玻片(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:AA00000112E00MNT10)
  8. Parafilm
  9. 棉绒
  10. 贴膜
  11. 安全手套
  12. Nigella damascena L。种子
  13. Click-iT EdU Alexa 555成像试剂盒(Thermo Fisher Scientific,Invitrogen TM,产品目录号:C10338)
  14. 胸苷(Sigma-Aldrich,目录号:T9250)

  15. 磷酸盐缓冲盐水(PBS),pH7.2(10x)(Thermo Fisher Scientific,Gibco TM,产品目录号:70013016)
  16. 乙醇
  17. 4',6-二脒基-2-苯基吲哚(DAPI)(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:62248)
  18. Tris基地
  19. 浓盐酸
  20. 多聚甲醛(Sigma-Aldrich,目录号:P6148)
  21. LR白色包埋试剂盒(Sigma-Aldrich,目录号:62662)
    注:此产品已停产。
  22. Formvar 1595E(Serva,目录号:21740)
  23. 无水1,2-二氯乙烷(Sigma-Aldrich,目录号:284505)
  24. Mowiol 4-88(Sigma-Aldrich,目录号:81381)
  25. 甘油(MP Biomedicals,目录号:04800687)
  26. 1,4-二氮杂双环 - [2.2.2] - 辛烷(Sigma-Aldrich,目录号:D2522)
    注:此产品已停产。
  27. 去离子H 2 O
  28. 1M Tris-HCl(pH8.5)(见配方1)
  29. 多聚甲醛(见方案2)
  30. LR白色丙烯酸树脂(见配方3)
  31. Formvar涂层盖玻片(见配方4)
  32. Mowion安装介质(见配方5)

设备

  1. 完美的循环(泰德佩拉,目录编号:13064)
  2. 轨道振荡器OS-20(Biosan,型号:OS-20,目录号:BS-010108-AAG)
  3. 实验室培养箱TC1 / 20(SKTB,型号:TC-1/20,目录号:1003)
  4. 化学通风橱
  5. Ultratome LKB III
  6. 配备ORCAII-ERG2相机(Hamamatsu)的荧光显微镜Axiovision 200M(Carl Zeiss,型号:Axiovision 200M)。
    注意:为了解卷积,使用了AxioVision 3.1软件(Carl Zeiss)。
  7. pH计
  8. 磁力搅拌器加热板MSH-300(Biosan,型号:MSH-300,目录号:BS-010302-OAA)
  9. 玻璃或钻石刀
  10. 微型离心机MiniSpin(Eppendorf,型号:MiniSpin®plus,目录号:5452000018)

程序

  1. 萌发Nigella damascena L的种子。在黑暗中25°С的90毫米培养皿中,用潮湿的滤纸覆盖。
    大约需要一个星期的时间才能长到10毫米
  2. 染色质标记,使用10毫米长的根。用50μMEdU(EdU是Click-iT EdU Alexa 555成像试剂盒的一部分,根据制造商的建议制备储备液)孵育发芽的种子30分钟。在25°C孵化器内的轨道摇床(50 rpm)上,用10 ml EdU溶液孵育90 mm培养皿。
  3. 在200μM胸腺嘧啶核苷中孵育种子30分钟,然后在不同的时间段内在蒸馏水中生长,以允许标记不同的染色体区域。在Nigella damascena L.的情况下,孵育3.5小时足以发现第一个标记的有丝分裂(Kuznetsova等人,2017)。在10毫升胸腺嘧啶核苷溶液或蒸馏水中孵育90毫米的陪替氏培养箱(50转每分钟)安装在一个孵化器在25°C。
  4. 使用单刃刀片从10mm长的根部切下长1.0mm的根尖,转移到1.5ml微管并在室温下在0.5xPBS中的2%多聚甲醛中固定1.5h(对于另外的固定方案参见注释1)。
    注意:使用化学通风橱处理含有甲醛的化学品。
  5. 固定后,在0.5倍PBS中清洗根尖,使用以下步骤使LR白色丙烯酸树脂脱水并渗透:
    1. 用0.5x PBS洗3次,每次5分钟。

    2. 用50%乙醇清洗根部30分钟
    3. 70%乙醇中的标本脱水2次,每次30分钟(可以在4°C下将标本储存在70%乙醇中)。

    4. 用LR White和70%乙醇(2:1)混合液(1 ml)渗透1小时。
      对于LR白色/乙醇混合物的制备,将一部分70%乙醇(逐滴加入)缓慢加入两份LR白色中,轻轻旋转试管(否则混合物将变成乳白色)。

    5. 在LR White(1 ml)中孵育1小时

    6. 在LR White(1 ml)中孵育过夜
    7. 在LR白色(1毫升)中孵育1小时。
    注意:除非另有说明,否则不要在室温下摇动。
  6. 在聚合发生时限制氧与树脂的接触是重要的。达到这个最方便的方法是使用明胶胶囊。将几根(3-4根)根放入胶囊的底部,用LR White填满边缘,然后滑动胶囊的另一半(图1)。


    图1.将根管从微管转移到明胶胶囊用于聚合。 为了从微管去除根部并转移到胶囊,使用直尖镊子是方便的,并且不会丢弃微管的LR White。
    用LR White填充明胶胶囊到边缘。


  7. 在55°C下聚合24小时
  8. 使用玻璃刀或钻石刀切割组织学样本,使用Ultratome准备半薄切片(100-250 nm)。

  9. 使用Perfect Loop在Formvar涂层封面纸上安装半薄切片(请参阅食谱)。
  10. 干燥的盖子在37°С的半薄切片滑动2-3小时,以更好地连接部分。
  11. 使用Click-iT EdU Alexa 555成像工具包根据制造商的协议检测EdU(见注2)。染色在黑暗中的潮湿室中进行。潮湿的房间是一个90毫米非通风的培养皿,其底部覆盖有石蜡膜。一块潮湿的棉花放在盘子墙附近。将一滴染料溶液置于石蜡膜上,将盖玻片置于这些滴剂上(部分向下)(图2)。这些盘子是用Parafilm密封的。


    图2.潮湿房间

  12. 在30 mm培养皿中用1μg/ ml DAPI溶液染色DNA 10 min,PBS洗5 min后,用抗漂白剂1,4-二氮杂双环[2.2.2] - 辛烷(DABCO)。
  13. 使用野外荧光显微镜和3D解卷积可以获取图像堆栈(图3)。


    图3.用EdU染色体标记。 Nigella damascena L的中期细胞。其染色体在晚期S期包括EdU。野外荧光显微镜与3D去卷积。比例尺= 5微米。

笔记

  1. 可以使用戊二醛固定(在50mMSörensen缓冲液中的2.5%戊二醛)。但是在这样的固定之后,苍白的自发荧光清晰可见。我们不能推荐这种传统光学显微镜的固定,但可以用于高分辨率显微镜和相关的光学和电子显微镜。
  2. 有许多来自不同制造商的荧光团叠氮化物(Molecular Probes,Lumiprobe,Sigma-Aldrich,等等)。用于染色的荧光染料的选择取决于用于图像获取的显微系统。我们的显微镜具有优化的检测Alexa 488和Alexa 555(或具有相似光谱特征的不同荧光染料)的滤光片组。我们更喜欢使用Alexa 555,因为我们使用这种荧光染料获得了更加明亮的染色。

食谱

  1. 1M Tris-HCl(pH8.5)
    1. 将Tris碱溶于水中(Tris碱121.14g,去离子水〜800ml)
    2. 使用pH计,用浓盐酸滴定溶液直至达到正确的pH值。
    3. 加去离子水至1000毫升
    4. 在4°C储存
  2. 多聚甲醛
    1. 将2.0g多聚甲醛与100ml0.5xPBS在玻璃中混合,玻璃用箔紧密封闭
    2. 用热板在磁力搅拌器上加热溶液直至透明(图4)。不要让解决方案煮沸。
      我们更喜欢用最高温度来加热
    3. 然后解决方案将是透明的,有必要从加热器中取出玻璃。稀释的甲醛可以等分,冷冻并储存在-20℃。我们从来没有储存等分超过1个月。每次解冻只有一次是很重要的
    注意:使用化学通风橱处理含有甲醛的化学品。


    图4.使用带热板的磁力搅拌器溶解多聚甲醛

  3. LR白色丙烯酸树脂
    1. 在室温下,将过氧化苯甲酰催化剂(1.998g)加入到LR白色丙烯酸树脂(100ml)中(两种试剂都是LR白色包埋试剂盒的一部分)
    2. 催化剂在室温下需要24小时才能完全溶解。在此期间,建议偶尔摇晃活化的树脂混合物。一旦与催化剂混合并充分溶解,树脂必须储存在2-8°C以保持其货架期但不超过9个月
    注意:在化学通风橱和安全手套下工作。如果您直接接触皮肤或眼睛,请用大量的水和肥皂清洗患处。
  4. Formvar涂层盖玻片
    将3毫克Formvar稀释在10毫升1,2-二氯乙烷中。这种解决方案可以存储在黑暗中的密封玻璃。清洁的盖玻片浸在Formvar的解决方案,并垂直干燥。 Formvar涂层的盖玻片应使用前准备
  5. Mowion安装介质
    1. 将1.2g Mowiol 4-88加入到3g甘油中,然后加入3ml去离子H 2 O并在室温下放置2小时。
    2. 加入6ml的0.2M Tris-HCl(pH8.5)并加热至50℃,偶尔混合直至Mowiol将被溶解。加入1,4-二氮杂双环[2,2,2] - 辛烷(DABCO)至终浓度2.5%以减少褪色
    3. 在离心机中以最大速度离心15分钟,将上清液转移到干净的微管中。
    4. 气密微型管中的等分试样可以储存在-20°C。

致谢

这项工作得到了俄罗斯科学基金会(项目14-15-00199)的支持。该协议是从以前的工作改编(库兹涅佐娃等人,2017年)。作者声明不存在可能影响本协议设计和实施的利益冲突或利益冲突。

参考

  1. Kuznetsova,M. A.,Chaban,I. A.和Sheval,E. V.(2017)。 可视化大染色体植物中的染色体浓缩 植物生物学< / 17>(1):153。
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引用:Sheval, E. V. (2018). Analysis of Chromosome Condensation/Decondensation During Mitosis by EdU Incorporation in Nigella damascena L. Seedling Roots. Bio-protocol 8(3): e2726. DOI: 10.21769/BioProtoc.2726.
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