搜索

Isolation of Rice Embryo Single Cell Type using Laser Capture Microdissection (LCM)
采用激光捕获显微切割(LCM)技术分离大米胚芽单细胞型   

下载 PDF 引用 收藏 提问与回复 分享您的反馈

本文章节

Abstract

A lot of transcriptional profiling in plant and animals has used RNAs samples from many different cell types. The laser-capture microdissection (LCM) can identify and harvest pure cellular populations directly from heterogenous tissues based on histological identification. The molecules or protein isolated from LCM-captured cells can be suitable for single cell type analysis by using chip expression profiling or sequencing.

Materials and Reagents

  1. Ethanol
  2. Acetic acid
  3. Histoclear (also named CitriSolv) (Thermo Fisher Scientific, catalog number: 5989-27-5 )
  4. DEPC H2O
  5. 75% (v/v) ethanol and 25% (v/v) acetic acid (see Recipes)
  6. Gradient series of ethanol solutions in H2O or histoclear (see Recipes)

Equipment

  1. Microscope
  2. Microtome (Waldorf, model: HM310 )
  3. Pix-Cell IIe LCM system (Arcturus)
  4. RNase-free glass slides
  5. 15 μm laser beam

Procedure

  1. Crytosectioning, fixation and dehydration of rice embryos
    1. Dissect rice embryo from the seeds under the microscope.
    2. Fix samples immediately on ice in fixation solution containing 75% (v/v) ethanol and 25% (v/v) acetic acid. The samples were left in the vials at 4 °C over night.
    3. Dehydrate the tissue in a series of ethanol concentrations (v/v) in order, 70%, 85%, 95%, 100%, each for 1 h at room temperature, followed by ethanol: histoclear (3:1, v/v), followed by ethanol: histoclear (1:1), ethanol: histoclear (1:3) and 100% histoclear treatment each for 1 h.
    4. Incubate the dehydrated samples in histoclear over night at 60 degree and then infiltrate with paraffin at 60 degree over 2 days, replacing histoclear with paraffin every 12 h.
    5. After embedding in paraffin, cut the embryo in 7 μm thick sections with a rotary microtome and place on RNase-free glass slides and store in darkness at 4 degree under dehydrating conditions with drierites.
    6. Deparaffinize sections in histoclear at room temperature for two changes of 10 min and air-dried before LCM.

  2. LCM
    1. Perform laser-capture microdissection using the Pix-Cell LCM system. After deparaffinizing and drying the tissues, laser-capture microdissect the interesting cells according to the manufacturer’s instructions.
    2. Based on the cell size diameters, the embryo organ cell types were isolated using 15 μm laser beam, laser power settings were 100 mW, and laser pulse durations were 2.5 ms. Embryo organ cell types were successfully identified and removed from heterogenous tissue by comparison of the difference among the images of the tissue before captured, the images of the tissue after removal of the harvested cells and the images of the cells captured on the cap. Generally, between 5 to 8 slides were processed each LCM caps and non-specific tissue were removed from the LCM cap using a Post-It note. See figure below.


Recipes

  1. 75% (v/v) ethanol and 25% (v/v) acetic acid
  2. Gradient series of ethanol solutions in H2O or histoclear

Acknowledgments

This protocol is adapted from Kerk et al. (2003) and Jiao et al. (2009).

References

  1. Jiao, Y., Tausta, S. L., Gandotra, N., Sun, N., Liu, T., Clay, N. K., Ceserani, T., Chen, M., Ma, L., Holford, M., Zhang, H. Y., Zhao, H., Deng, X. W. and Nelson, T. (2009). A transcriptome atlas of rice cell types uncovers cellular, functional and developmental hierarchies. Nat Genet 41(2): 258-263.
  2. Kerk, N. M., Ceserani, T., Tausta, S. L., Sussex, I. M. and Nelson, T. M. (2003). Laser capture microdissection of cells from plant tissues. Plant Physiol 132(1): 27-35.
  3. Nelson, T., Tausta, S. L., Gandotra, N., and Liu, T. (2006). Laser microdissection of plant tissue: what you see is what you get. Annu Rev Plant Biol 57: 181-201.

简介

植物和动物中的许多转录谱使用来自许多不同细胞类型的RNA样品。 激光捕获显微解剖(LCM)可以基于组织学鉴定直接从异源组织中鉴定和收获纯细胞群体。 从LCM捕获的细胞分离的分子或蛋白可以适合于通过使用芯片表达谱或测序的单细胞类型分析。

材料和试剂

  1. 乙醇
  2. 乙酸
  3. Histoclear(也称为CitriSolv)(Thermo Fisher Scientific,目录号:5989-27-5)
  4. DEPC H 2 O
  5. 75%(v/v)乙醇和25%(v/v)乙酸(参见Recipes)
  6. 在H 2 O或组织清蛋白中的乙醇溶液的梯度系列(参见配方)

设备

  1. 显微镜
  2. 切片机(Waldorf,型号:HM310)
  3. Pix-Cell IIe LCM系统(Arcturus)
  4. 无核糖酶玻璃载玻片
  5. 15μm激光束

程序

  1. 水稻胚胎的冷冻保护,固定和脱水
    1. 从显微镜下的种子解剖水稻胚胎。
    2. 将样品立即固定在含有75%(v/v)乙醇和25%(v/v)乙酸的固定溶液中的冰上。 将样品在4℃下放置在小瓶中过夜。
    3. 在一系列乙醇浓度(v/v)中依次将组织脱水70%,85%,95%,100%,在室温下各洗涤1小时,接着用乙醇:组织清洗(3:1,v/v ),然后用乙醇:组织清蛋白(1:1),乙醇:组织清蛋白(1:3)和100%组织清蛋白处理1小时。
    4. 孵育脱水的样品在组织培养物中过夜,60度,然后用石蜡60度浸润2天,每12小时用石蜡替代组织透明液。
    5. 在石蜡包埋后,用旋转切片机切成7μm厚切片的胚,并放置在无核糖酶的玻璃载玻片上,并在具有干燥剂的脱水条件下在4℃的黑暗中储存。
    6. 将组织切片中的切片在室温下进行两次变化10分钟,并在LCM之前风干。

  2. LCM
    1. 使用Pix-Cell LCM系统进行激光捕获显微切割。脱蜡和干燥组织后,激光捕获显微解剖感兴趣的细胞,根据制造商的说明。
    2. 基于细胞大小直径,使用15μm激光束分离胚器官细胞类型,激光功率设置为100mW,并且激光脉冲持续时间为2.5ms。通过比较捕获之前组织的图像之间的差异,移除收获的细胞后的组织的图像和捕获在帽上的细胞的图像,成功地鉴定了胚器官细胞类型并从异源组织中除去。通常,每个LCM帽处理5至8个载玻片,使用Post-It注释从LCM帽除去非特异性组织。见下图。


食谱

  1. 75%(v/v)乙醇和25%(v/v)乙酸
  2. 梯度系列H 2 O或乙醇溶液中的乙醇溶液

致谢

该协议改编自Kerk等人(2003)和Jiao等人(2009)。

参考文献

  1. Jiao,Y.,Tausta,S.L.,Gandotra,N.,Sun,N.,Liu,T.,Clay, Ceserani,T.,Chen,M.,Ma,L.,Holford,M.,Zhang,H.Y.,Zhao, Deng,X.W。和Nelson,T。(2009)。 水稻细胞类型的转录组图谱揭示细胞,功能和发育层次。 Nat Genet 41(2):258-263。
  2. Kerk,N.M.,Ceserani,T.,Tausta,S.L.,Sussex,I.M.and Nelson,T.M。(2003)。 从植物组织激光捕获显微解剖细胞。 植物生理学 132(1):27-35。
  3. Nelson,T.,Tausta,S.L.,Gandotra,N.,and Liu,T。(2006)。 植物组织的激光显微切割:您所看到的是您所获得的。 Annu Rev Plant Biol 57:181-201
  • 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: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Liu, T. (2012). Isolation of Rice Embryo Single Cell Type using Laser Capture Microdissection (LCM) . Bio-protocol 2(18): e259. DOI: 10.21769/BioProtoc.259.
提问与回复

(提问前,请先登录)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。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。