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Plastic Embedding and Sectioning of Plant Tissues
植物组织树脂包埋与切片   

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参见作者原研究论文

本实验方案简略版
Micron
Feb 2011

Abstract

Plastic (resin) embedding provides exclusively improvements to cellular definition compared to paraffin embedding. The combination of strongly cross-linking paraformaldehyde with glutaraldehyde and post fixed with OsO4 is the fixative of choice for high-resolution light microscopy and electron microscopy. For this reason, this method is an ideal tool for visualizing plant cellular morphology and phenotype.

Materials and Reagents

  1. Ethanol
  2. Acetone
  3. Na2HPO4
  4. Gelatin capsules (Electron Microscopy Sciences, catalog number: 71012 )
  5. Glutaraldehyde (Fluka, catalog number: 49627 )
  6. Paraformaldehyde
  7. OsO4
  8. Catalyst
  9. Osmium (Fluka, catalog number: 75633 )
  10. Fixative solution (see Recipes)
  11. Post-fixative solution (see Recipes) 
  12. NaPO4 buffer (see Recipes)
  13. Resin (LR white) (Fluka, catalog number: 62662 ) (see Recipes) 

Equipment

  1. Shaker
  2. Incubator with temperature control
  3. Light or electron microscopy
  4. Vacuum
  5. Glass blade or jeweler saw (Electron Microscopy Sciences, catalog number: 71012 )

Procedure

  1. Fixation: Cut and fix plant tissues in fixative solution for 2 h, then vacuum for 15 min without shaking. If the tissues did not sink, re-infiltrated for another 10 min. The tissues can be stored at 4 °C over night.
  2. Rinse: Rinse the entire samples in 0.5 M NaPO4 buffer, and then wash tissues in 0.5 M NaPO4 buffer for 3 times; 15 min each at 4 °C. Add enough NaPO4 buffer to cover the entire tissues.
  3. Post-fix: Transfer samples to 1% Osmium (OsO4) in 0.5 M NaPO4 buffer for 1 h in the dark. The samples should be immersed in the post-fixative solution. The tissues can be kept for another hour in post-fixative solution until they all turned black.
  4. Wash without changing container: 3 times in ddH2O; 10 min each at 4 °C on a rotating shaker.
  5. Dehydration without shaking: Dehydrate the tissues in a graded ethanol series as below:
    12.5% ethanol, 10 min
    25% ethanol, 10 min
    35% ethanol, 10 min
    50% ethanol, 10 min
    70% ethanol, 10 min
    80% ethanol, 10 min
    95% ethanol, 10 min
    100% ethanol, 10 min (twice)
    100% acetone, 10min (twice)
  6. Infiltration: To damp the old solution and exchange the 100% ethanol with acetone-resin mixture as following steps:
    Acetone: resin (1:1) 1 h
    Acetone: resin (1:2) 1/2 h
    100% resin 1 h
    100% resin over night on shaker
  7. Polymerize: Put samples in gelatin capsules filled with 100% fresh resin, leave samples at 50 °C for 60 h. Once the resin was solidified, the capsules can be kept at 4 °C for a few weeks.
  8. Blocks can be trimmed and cut with glass blade or jewelers saw depending on what shapes were required for light or electron microscopy. To position the blocks depend on the dimension (cross, transverse, longitudinal) section layout was required. The sections were picked up and floated out on 30% acetone on a warmer plate (42 °C) until dry.

Recipes

  1. Fixative solution (make fresh with ddH2O)
    3% Glutaraldehyde (10% stock) 
    1.5% Paraformaldehyde (16% stock) 
    1.6% 1 M NaPO4 
  2. Post-fixative solution (make fresh)
    OsO4 (4% stock)   2 ml
    1 M NaPO4           4 ml
    H2O                      2 ml
  3. 1 M NaPO4 buffer (pH 6.8)
    NaH2PO4.H2O     137.99 g/L
    Na2HPO4            141.97 g/L
  4. Resin (LR white)
    Add 9.9 g catalyst to 500 ml LR white, shaken thoroughly.

Acknowledgments

This protocol is adapted from Alpers and Beckstead (1985) and Massover (2011).

References

  1. Alpers, C. E. and Beckstead, J. H. (1985). Enzyme histochemistry in plastic-embedded sections of normal and diseased kidneys. Am J Clin Pathol 83(5): 605-612.
  2. Massover, W. H. (2011). New and unconventional approaches for advancing resolution in biological transmission electron microscopy by improving macromolecular specimen preparation and preservation. Micron 42(2): 141-151.

简介

与石蜡包埋相比,塑料(树脂)包埋仅提供细胞定义。 强交联的多聚甲醛与戊二醛的组合以及用OsO 4后固定的组合是用于高分辨率光学显微镜和电子显微镜的固定剂。 因此,这种方法是可视化植物细胞形态和表型的理想工具。

材料和试剂

  1. 乙醇
  2. 丙酮
  3. Na HPO 4
  4. 明胶胶囊(Electron Microscopy Sciences,目录号:71012)
  5. 戊二醛(Fluka,目录号:49627)
  6. 多聚甲醛
  7. OsO 4
  8. 催化剂
  9. 锇(Fluka,目录号:75633)
  10. 固定溶液(见配方)
  11. 后固定溶液(见配方)
  12. NaPO <4>缓冲液(参见配方)
  13. 树脂(LR白色)(Fluka,目录号:62662)(参见Recipes)

设备

  1. 振动器
  2. 带温度控制的孵育器
  3. 光或电子显微镜
  4. 真空
  5. 玻璃刀片或珠宝锯(Electron Microscopy Sciences,目录号:71012)

程序

  1. 固定:在固定溶液中剪切并固定植物组织2小时,然后真空15分钟,不摇动。 如果组织没有下沉,再渗透另外10分钟。 组织可以在4℃下储存过夜。
  2. 冲洗:在0.5M NaPO 4缓冲液中冲洗整个样品,然后在0.5M NaPO 4缓冲液中洗涤组织3次; 在4℃下各15分钟。 加入足够的NaPO 4缓冲液以覆盖整个组织
  3. 后固定:在黑暗中将样品转移到在0.5M NaPO 4缓冲液中的1%锇(OsO 4)中1小时。 样品应浸入后固定溶液中。 组织可以在后固定溶液中保持另一小时,直到它们全部变黑
  4. 洗涤时不更换容器:在ddH 2 O中3次; 在旋转振荡器上在4℃下各10分钟。
  5. 脱水不摇动:在如下分级乙醇系列中脱水组织:
    12.5%乙醇,10分钟
    25%乙醇,10分钟
    35%乙醇,10分钟
    50%乙醇,10分钟
    70%乙醇,10分钟
    80%乙醇,10分钟
    95%乙醇,10分钟
    100%乙醇,10分钟(两次) 100%丙酮,10分钟(两次)
  6. 渗透:为了衰减旧溶液并用丙酮 - 树脂混合物交换100%乙醇,步骤如下:
    丙酮:树脂(1:1)1 h
    丙酮:树脂(1:2)1/2小时
    100%树脂1 h
    100%树脂,在振动器上超过
  7. 聚合:将样品放入填充有100%新鲜树脂的明胶胶囊中,将样品在50℃下放置60小时。 一旦树脂固化,胶囊可以在4℃保持几周
  8. 块可以修剪和切割用玻璃刀片或珠宝锯,取决于什么形状需要光或电子显微镜。 为了定位块取决于尺寸(横向,横向,纵向)部分布局是必需的。 取出切片并在温热板(42℃)上漂浮在30%丙酮上直到干燥

食谱

  1. 固定溶液(用ddH 2 O制成新鲜溶液)
    3%戊二醛(10%股票) 
    1.5%多聚甲醛(16%储备液) 
    1.6%1 M NaPO 4
  2. 后固定溶液(使新鲜)
    OsO <4>(4%股票)  2 ml
    1 M  NaPO <4> 4           4 ml
    H 2 O                      2 ml
  3. 1 M NaPO 4缓冲液(pH 6.8) NaH 2 2 PO 4 .H 2 O 2。   137.99 g/L
    Na 2 HPO <4>            141.97 g/L
  4. 树脂(LR白色)
    加入9.9g催化剂至500ml LR白色,充分摇动。

致谢

该协议改编自Alpers和Beckstead(1985)和Massover(2011)。

参考文献

  1. Alpers,C.E。和Beckstead,J.H。(1985)。 正常和患病肾脏的塑料包埋切片中的酶组织化学。 J Clin Pathol 83(5):605-612。
  2. Massover,W.H。(2011)。 通过改进生物透射电子显微镜技术提高分辨率的新的和非常规的方法 大分子标本制备和保存。 42(2):141-151。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Liu, T. (2012). Plastic Embedding and Sectioning of Plant Tissues. Bio-protocol 2(23): e296. DOI: 10.21769/BioProtoc.296.
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