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Thin Sections of Technovit 7100 Resin of Rice Endosperm and Staining
采用Technovit 7100树脂制备水稻胚乳薄切片并染色   

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Abstract

Starch is a biologically and commercially important carbohydrate that is accumulated in plant storage organs, such as seed endosperm. Starch is water-insoluble and forms transparent grains, referred to as starch grains (SGs). SGs are easily stained by iodine solution and can be observed under a normal microscope. Technovit 7100 resin is suitable for preparation of thin sections from endosperm. The thin sections and iodine staining can visualize SGs clearly inside the endosperm cell. This protocol provides the procedures to prepare thin sections of Technovit 7100 resin from rice endosperm. It can also be applicable to the seeds of other plant species.


Figure 1. Iodine-stained Technovit thin sections of matured endosperm cells in rice and barley. Starch grains (SGs) can be visualized as violet particles.

Materials and Reagents

  1. Rice seeds
  2. Technovit 7100 resin (Heraeus Kulzer)
  3. Lugol solution (MP Biomedicals, catalog number: 155269 )
  4. Formalin
  5. Acetic acid
  6. 99.5% ethanol (commercially available one)
  7. Instant adhesives (Aron alpha, Toagosei Co.)
  8. FAA solution (see Recipes)
  9. 100% ethanol (see Recipes)
  10. 100% Technovit 7100 (I) (see Recipes)
  11. 25% Technovit 7100 resin (I) (see Recipes)
  12. 50% Technovit 7100 resin (I) (see Recipes)
  13. 75% Technovit 7100 resin (I) (see Recipes)
  14. 100% Technovit 7100 (II) (see Recipes)

Equipment

  1. Silanized slides (Dako, catalog number: S3003 )
  2. Wooden dowels (6 x 25 mm)
  3. Razor blade (Feather, catalog number: FH-10 )
  4. 1.5 ml plastic tube
  5. 0.2 ml PCR tube
  6. Molecular sieves 4A 1/16 (Nacalai tesque, catalog number: 04172-95 )
  7. Ultramicrotome (Leica Microsystems, model: EM UC7 )
  8. Diamond knife (Diatome AG, Ultra trim, 3.0 mm)
  9. Rotator (TAITEC, model: RT-30mini )
  10. Forceps (Vigor, stainless steel #5)
  11. Aspirator (Ulvac, model: MDA-015 )
  12. Microscope (OLYMPUS, model: AX70 )


Figure 2. A wooden dowel

Procedure

  1. Resin embedding of endosperm
    1. Cut out approximately 1 mm cubic blocks from the center region of the endosperm in brown rice seeds using razor blades.
    2. Put the blocks into an empty 1.5-ml plastic tube.
    3. Add 500 µl of FAA solution into the plastic tube. The blocks should be sunken at the bottom of tube.
    4. Make the tube open and place it in the aspirator and vacuum for 5 min.
    5. Release the negative pressure gently.
    6. Repeat steps 4 and 5 two more time.
    7. Take the tube from the aspirator and remove the FAA by micropipette.
    8. Add 1 ml of fresh FAA solution into the tube.
    9. Rotate at 30 r/min overnight at room temperature.
    10. Remove FAA and add 1 ml of 30% ethanol.
    11. Rotate at 30 r/min for 1 h at room temperature.
    12. Remove 30% ethanol and add 1 ml of 50% ethanol.
    13. Rotate at 30 r/min for 1 h at room temperature.
    14. Remove 50% ethanol and add 1 ml of 70% ethanol.
    15. 15. Rotate at 30 r/min for 1 h at room temperature.
    16. Remove 70% ethanol and add 1 ml of 90% ethanol.
    17. Rotate at 30 r/min for 1 h at room temperature.
    18. Remove 90% ethanol and add 1 ml of 95% ethanol.
    19. Rotate at 30 r/min for 1 h at room temperature.
    20. Remove 95% ethanol and add 1 ml of 100% ethanol.
    21. Rotate at 30 r/min for 1 h at room temperature.
    22. Exchange 100% ethanol and rotate at 30 r/min for 1 h.
    23. Exchange 100% ethanol and rotate at 30 r/min overnight at room temperature.
    24. Exchange 100% ethanol and rotate at 30 r/min for 1 h at room temperature.
    25. Remove 100% ethanol and add 1 ml of 25% Technovit 7100 resin (I) Rotate at 30 r/min for 1 h at room temperature.
    26. Remove 25% Technovit 7100 resin (I) and add 1 mL of 50% Technovit 7100 resin (I).
    27. Rotate at 30 r/min for 1 h at room temperature.
    28. Remove 50% Technovit 7100 resin (I) and add 1 ml of 75% Technovit 7100 resin (I).
    29. Rotate at 30 r/min for 1 h at room temperature.
    30. Remove 75% Technovit 7100 resin (I) and add 1 ml of 100% Technovit 7100 resin (I).
    31. Rotate at 30 r/min for 1 h at room temperature.
    32. Exchange 100% Technovit 7100 resin (I) and rotate at 30 r/min for 1 h at room temperature.
    33. Exchange 100% Technovit 7100 resin (I) and rotate at 30 r/min overnight at room temperature.
    34. Exchange 100% Technovit 7100 resin (I) and rotate at 30 r/min for 1 h at room temperature.
    35. Remove 100% Technovit 7100 resin (I) from the tube at room temperature.
    36. Transfer the endosperm block into the 0.2-ml PCR tube. One PCR tubes should contain one endosperm block.
    37. Add 150-200 µl of 100% Technovit 710 (II) in the PCR tube.
    38. Confirm that the endosperm block precipitate on the bottom of the PCR tube. If floated, try to send to the bottom by pipetting.
    39. Incubate the PCR-tube at 37 °C for more than 2 day to solidify the resin.

  2. Trimming the solidified resin
    1. Remove the PCR tube from the solidified resin by razor blades.
    2. Trim the solidified resin by razor blades to remove the extra resin. Leave intact the part where the endosperm block is embedded.
    3. Adhere the trimmed resin on the wooden dowel by instant super adhesives.

  3. Thin sectioning and staining
    1. Fix the wooden dowel on the sample holder of ultramicrotome to make the resin facing outward.
    2. Set the diamond knife on the ultramicrotome.
    3. Adjust the angles, directions and positions of the dowel and diamond knife to make the knife-edge can approach at appropriate directions to the endosperm blocks in the resin.
    4. Start sectioning of resin by preceding the position of diamond knife by 1 µm each.
    5. Continue the sectioning of resin to obtain the thin sections containing endosperm blocks.
    6. Prepare a drop of water on a silanized slide.
    7. Pick up the thin section containing endosperm blocks by forceps.
    8. Put the thin section onto the surface of water drop on the slide. The thin section will expand on the surface of water.
    9. Leave the thin section overnight at room temperature to dry out the water.
    10. Add a drop of 40-times diluted Lugol solution on the dried thin section stuck on the slide.
      Note: It takes 1-5 sec to stain the sections. And there is no need  to remove the dye before observation under microscope.
    11. Mount a coverglass on the stained section.
      Note: No special mounting reagent is needed. The rest of the dye works as the mounting reagent.
    12. Examine the section under a normal light microscope.

Recipes

  1. FAA solution
    1 ml formalin
    1 ml acetic acid
    9 ml 99.5% ethanol
    9 ml H2O
  2. 100% ethanol
    500 ml ethanol (99.5%, commercially available) approximately 100 g molecular sieves 4A 1/16
  3. 100% Technovit 7100 (I)
    10 ml Technovit 7100
    0.1 g Hardener I
  4. 25% Technovit 7100 resin (I)
    250 µl 100% Technovit 7100 (I)
    750 µl 100% ethanol
  5. 50% Technovit 7100 resin (I)
    500 µl 100% Technovit 7100 (I)
    500 µl 100% ethanol
  6. 75% Technovit 7100 resin (I)
    750 µl 100% Technovit 7100 (I)
    250 µl 100% ethanol
  7. 100% Technovit 7100 (II)
    1 ml Technovit 7100 (I)
    66 µl Hardener II

Acknowledgments

This work was funded by the Ministry of Education, Culture, Sports, Science and Technology Grant-in-Aid for Scientific Research (no. 23770046). This work was also supported by the following foundation; the Iijima Memorial Foundation for the Promotion of Food Science and Technology, the Japan Prize Foundation, Shorai Foundation for Science and Technology, Wesco Scientific Promotion Foundation, The Towa Foundation for Food Research, the Foundation of Skylark Food Science Institute and Oohara Foundation.

References

  1. Matsushima, R., Maekawa, M., Fujita, N. and Sakamoto, W. (2010). A rapid, direct observation method to isolate mutants with defects in starch grain morphology in rice. Plant Cell Physiol 51(5): 728-741.
  2. Matsushima, R., Yamashita, J., Kariyama, S., Enomoto, T., Sakamoto, W. (2013). A phylogenetic re-evaluation of morphological variations of starch grains among Poaceae species. J Applied Glycoscience 60:37-44.
  3. Matsushima, R., Maekawa, M., Kusano, M., Kondo, H., Fujita, N., Kawagoe, Y. and Sakamoto, W. (2014). Amyloplast-localized SUBSTANDARD STARCH GRAIN4 protein influences the size of starch grains in rice endosperm. Plant Physiol 164(2): 623-636.

简介

淀粉是在植物储存器官(例如种子胚乳)中积累的生物学上和商业上重要的碳水化合物。 淀粉是水不溶性的并且形成称为淀粉颗粒(SGs)的透明颗粒。 SGs容易被碘溶液染色,并且可以在正常显微镜下观察。 Technovit 7100树脂适用于从胚乳制备薄切片。 薄切片和碘染色可以明显地在胚乳细胞内部显现SG。 该协议提供了从水稻胚乳制备Technovit 7100树脂薄片的程序。 它也可以适用于其他植物物种的种子。


淀粉颗粒(SG)可以被视为紫色颗粒。

材料和试剂

  1. 水稻种子
  2. Technovit 7100树脂(Heraeus Kulzer)
  3. Lugol溶液(MP Biomedicals,目录号:155269)
  4. 福尔马林
  5. 乙酸
  6. 99.5%乙醇(可商购的)
  7. 速溶胶粘剂(Aron alpha,Toagosei Co.)
  8. FAA解决方案(参见配方)
  9. 100%乙醇(见配方)
  10. 100%Technovit 7100(I)(见配方)
  11. 25%Technovit 7100树脂(I)(参见配方)
  12. 50%Technovit 7100树脂(I)(参见配方)
  13. 75%Technovit 7100树脂(I)(参见配方)
  14. 100%Technovit 7100(II)(见配方)

设备

  1. 硅烷化载玻片(Dako,目录号:S3003)
  2. 木钉(6 x 25毫米)
  3. 剃刀刀片(羽毛,目录号:FH-10)
  4. 1.5 ml塑料管
  5. 0.2 ml PCR管
  6. 分子筛4A 1/16(Nacalai tesque,目录号:04172-95)
  7. 超微切片机(Leica Microsystems,型号:EM UC7)
  8. 金刚石刀(Diatome AG,Ultra trim,3.0mm)
  9. 旋转器(TAITEC,型号:RT-30mini)
  10. 钳(Vigor,不锈钢#5)
  11. 抽吸器(Ulvac,型号:MDA-015)
  12. 显微镜(OLYMPUS,型号:AX70)


图2.木榫

程序

  1. 胚乳的树脂包埋
    1. 使用刀片从糙米种子胚乳的中心区域切出大约1毫米立方体块
    2. 将块放入一个空的1.5毫升塑料管。
    3. 加入500μl的FAA溶液到塑料管中。 块应该在管的底部下沉。
    4. 使管打开,将其放置在吸气器中并真空5分钟。
    5. 轻轻释放负压。
    6. 重复步骤4和5两次以上。
    7. 从吸气器取管,用微量移液管取出FAA
    8. 将1ml新鲜的FAA溶液加入试管中
    9. 在室温下以30转/分钟旋转过夜
    10. 取出FAA,加入1ml 30%乙醇
    11. 在室温下以30转/分钟旋转1小时
    12. 除去30%乙醇,加入1ml 50%乙醇
    13. 在室温下以30转/分钟旋转1小时
    14. 除去50%乙醇,加入1ml 70%乙醇
    15. 15.在室温下以30转/分钟旋转1小时
    16. 除去70%乙醇,加入1ml 90%乙醇
    17. 在室温下以30转/分钟旋转1小时
    18. 除去90%乙醇,加入1ml 95%乙醇
    19. 在室温下以30转/分钟旋转1小时
    20. 除去95%乙醇,加入1ml 100%乙醇
    21. 在室温下以30转/分钟旋转1小时
    22. 交换100%乙醇,并以30转/分钟旋转1小时
    23. 交换100%乙醇,并在室温下以30转/分钟旋转过夜
    24. 交换100%乙醇,在室温下以30转/分钟旋转1小时
    25. 除去100%乙醇,加入1ml 25%的Technovit 7100树脂(I)在室温下以30转/分钟旋转1小时。
    26. 除去25%Technovit 7100树脂(I),并加入1mL 50%Technovit 7100树脂(I)。
    27. 在室温下以30转/分钟旋转1小时
    28. 除去50%Technovit 7100树脂(I),并加入1ml 75%Technovit 7100树脂(I)。
    29. 在室温下以30转/分钟旋转1小时
    30. 除去75%Technovit 7100树脂(I),并加入1ml 100%Technovit 7100树脂(I)。
    31. 在室温下以30转/分钟旋转1小时
    32. 交换100%Technovit 7100树脂(I)并在室温下以30转/分钟旋转1小时
    33. 交换100%Technovit 7100树脂(I)并在室温下以30转/分钟旋转过夜
    34. 交换100%Technovit 7100树脂(I)并在室温下以30转/分钟旋转1小时
    35. 在室温下从管中除去100%Technovit 7100树脂(I)
    36. 将胚乳块转移到0.2-ml PCR管中。 一个PCR管应该包含一个胚乳块
    37. 在PCR管中加入150-200μl的100%Technovit 710(II)
    38. 确认胚乳块沉淀在PCR管的底部。 如果漂浮,尝试通过移液器发送到底部。
    39. 在37℃下孵育PCR管2天以上以固化树脂

  2. 修整固化树脂
    1. 用刀片从固化的树脂上取下PCR管
    2. 用刀片修整固化的树脂,以除去额外的树脂。 保持胚乳块嵌入的部分完好无损。
    3. 通过瞬间超级粘合剂将修剪的树脂粘在木销钉上。

  3. 薄切片和染色
    1. 将木钉固定在超薄切片机的样品架上,使树脂朝外。
    2. 将菱形刀放在超薄切片机上。
    3. 调整榫钉和金刚石刀的角度,方向和位置,使刀口能够在适当的方向接近树脂中的胚乳块。
    4. 通过在每个金刚石刀的位置前1微米开始树脂切片
    5. 继续切片树脂以获得含有胚乳块的薄切片
    6. 在硅烷化的载玻片上准备一滴水。
    7. 用镊子取出含有胚乳块的薄切片
    8. 将薄片放在幻灯片上的水滴表面。 薄部分将在水面上膨胀。
    9. 将薄片在室温下放置过夜以干燥水。
    10. 在粘在载玻片上的干薄片上加入一滴40倍稀释的Lugol溶液。
      注意:染色部分需要1-5秒。 而且没有必要 以在显微镜下观察之前除去染料。
    11. 在染色部分装上盖玻片。
      注意:不需要特殊的安装试剂。 其余的染料作为固定试剂。
    12. 在正常光学显微镜下检查切片。

食谱

  1. FAA解决方案
    1ml福尔马林
    1ml乙酸 9ml 99.5%乙醇 9ml H 2 O 2 /
  2. 100%乙醇
    500ml乙醇(99.5%,市售)约100g分子筛4A 1/16
  3. 100%Technovit 7100(I)
    10 ml Technovit 7100
    0.1g硬化剂I
  4. 25%Technovit 7100树脂(I)
    250μl100%Technovit 7100(I)
    750μl100%乙醇
  5. 50%Technovit 7100树脂(I) 500μl100%Technovit 7100(I)
    500μl100%乙醇
  6. 75%Technovit 7100树脂(I) 750μl100%Technovit 7100(I)
    250μl100%乙醇
  7. 100%Technovit 7100(II)
    1 ml Technovit 7100(I)
    66μl固化剂II

致谢

这项工作由教育,文化,体育,科学和技术促进科学研究资助部(第23770046号)资助。这项工作也得到以下基础的支持: Iijima纪念基金会,日本奖基金会,Shorai科学技术基金会,Wesco科学促进基金会,Towa食品研究基金会,Skylark食品科学研究所基金会和Oohara基金会。

参考文献

  1. Matsushima,R.,Maekawa,M.,Fujita,N.and Sakamoto,W。(2010)。 一种快速,直接的观察方法,用于分离具有水稻淀粉颗粒形态缺陷的突变体。 Plant Cell Physiol 51(5):728-741
  2. Matsushima,R.,Yamashita,J.,Kariyama,S.,Enomoto,T.,Sakamoto,W。(2013)。 系统发育学重新评估形态学变异 应用糖科学 60:37-44。
  3. Matsushima,R.,Maekawa,M.,Kusano,M.,Kondo,H.,Fujita,N.,Kawagoe,Y.and Sakamoto,W.(2014)。 淀粉体本地化的SUBSTANDARD STARCH GRAIN4蛋白影响水稻胚乳中淀粉颗粒的大小。 植物生理学 164(2):623-636。
  • 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。
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Matsushima, R. (2014). Thin Sections of Technovit 7100 Resin of Rice Endosperm and Staining. Bio-protocol 4(18): e1239. DOI: 10.21769/BioProtoc.1239.
  2. Matsushima, R., Maekawa, M., Kusano, M., Kondo, H., Fujita, N., Kawagoe, Y. and Sakamoto, W. (2014). Amyloplast-localized SUBSTANDARD STARCH GRAIN4 protein influences the size of starch grains in rice endosperm. Plant Physiol 164(2): 623-636.
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