薄切片进行免疫组化切片的筹备工作

Published 4/28/2007
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Biology

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Summary

本方法可重复性的低温恒温器的小,难以管理,组织块,如活组织切片检查和脑切片,切片。我们利用一个简单的铝冻结的阶段,以方便处理组织和一个标准的低温​​恒温器,定期制作5-10微米,400微米厚的脑片连续切片。

Cite this Article

Copy Citation

Park, J., Cunningham, M. G. Thin Sectioning of Slice Preparations for Immunohistochemistry. J. Vis. Exp. (3), e194, doi:10.3791/194 (2007).

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Abstract

在神经科学,以及其他学科的许多调查,参与研究的小,但新鲜宏观件或部分组织已被保存下来,删除,或切除,但保持可行的,在脑组织切片准备。随后的微观研究,这种材料可以是具有挑战性,组织样本可能难以处理。证明这里是一个薄低温恒温器的部分,厚度为0.2-5.0毫米的范围可以从组织获得的方法。我们经常削减400微米厚Vibratome的脑片串联成5-10微米日冕低温恒温器的部分。切片通常首先用于电生理实验,然后需要的地区,从录音,观察细胞结构的微观分析。我们已经构建了一个简单的装置,可控制和可重复性的准备和组织切片定位。该装置由一个圆柱体5厘米的长度,用直径1.2厘米,作为冻结切片阶段。一环,紧贴幻灯片在气缸周围墙壁,让沉浸在冻结复合组织(例如,华侨城)切片。然后迅速冷冻粉碎的干冰和由此产生的晶圆可低温恒温切片的位置容易。薄片可以解冻安装在涂幻灯片,以便进一步的研究来进行,如各种染色方法,原位杂交,免疫组织化学,这里展示。

Protocol

  1. 准备从磁带华侨城平台的模具。
  2. 与华侨城填充模具。冻结范围内的低温恒温器或通过使用粉碎的干冰。
  3. 删除冻结华侨城平台周围的磁带。
  4. 将冻结夹头和低温恒温器的安装阶段,并锁定在夹头的标记。
  5. 组通过OCT平台,直到表面是平坦的。
  6. 低温恒温器冻结阶段,取出重铺华侨城平台和地点。
  7. 华侨城广场组织样本(以前用30%甘油或蔗糖的PBS冻存)。
  8. 准备冻结列外圈形成华侨城以及列的顶部以上约5毫米投影。
  9. 小心冻结柱表面的中心位置上组织样本,并慢慢加入华侨城,直到充满。
  10. 环绕粉碎干冰冻结列。组织和OCT应完全冻结在20-60秒内。
  11. 准备在温度的增加,外圈可去除样品仍然冻结。
  12. 滑出冻结列侧身的样品,在低温恒温器的地方。
  13. 华侨城华侨城平台和位置的标本(组织下来)申请公司的压力表面放置下降。试样迅速冻结到华侨城平台。
  14. 安全夹头到低温恒温器的安装与对齐标记阶段。
  15. 通过华侨城第肤浅的组织标本。
  16. 解冻安装到玻片薄片和冻结或在室温下储存。
  17. 免疫反应可以在玻片上的组织。
  18. 试剂汇集到幻灯片,可以轻轻地激动,如果光敏感,可能会覆盖。
  19. 以后各阶段的反应很容易进行反相幻灯片浪费插座,吸湿排汗的幻灯片,然后应用在未来试剂。

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Discussion

这里介绍的协议为研究人员提供一个简洁,易于遵循大纲如何获得小,难以管理,组织块,如活检和大脑切片进行进一步研究,如薄的低温恒温器部分各种染色方法,原位杂交,免疫组织化学。

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Materials

Name Company Catalog Number Comments
O. C. T. Ted Pella, Inc. 27050
Glycerol Solution 30% Glycerol Solution in PBS w/v
Sucrose Solution 30% Sucrose Solution in PBS w/v

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References

  1. Scoutern, C. W., O'Connor, R., Cunningham, M. Perfusion fixation of research animals. Microsc. Today. 14, 26-33 (2006).
  2. Cunningham, M. G., Connor, C. M., Zhang, K., Benes, F. M. Diminished serotonergic innervation of adult medial prefrontal cortex after 6-OHDA lesions in the newborn rat. Brain Res. Dev. Brain Res. 157, 124-131 (2005).

Comments

3 Comments

  1. Thank-you for posting this video. Your device seems to work well with small tissue, but what do you use for freezing whole adult rat brains? Is the powdered dry ice method sufficient alone to reduce freezing artifact if you are freezing a whole adult brain? Do you have an alternative procedure for these applications?   Much appreciated,
    Neil

    Reply
    Posted by: Anonymous
    October 23, 2008 - 4:04 AM
  2. Hi Neil, you actually can get a pretty good freeze with finely powdered dry ice. Just cover and let freeze for 3-5 minutes.  I would recommend, however, immersing the whole brain in isopentane that has been chilled (~30 minutes) in dry ice.  Just leave the brain in isopentane for 30 seconds, otherwise it can distort and fracture.  You will then need to let the (very cold, -80 degrees) brain equilibrate (warm up) to your cryostat or microtome temperature in order for it to cut smoothly.
    Good luck!
    Miles

    Miles G. Cunningham, MD PhD
    Director, Laboratory for Neural Reconstruction
    Administrative Director, McLean Fellowship in
    Neuropsychiatry and Behavioral Neurology
    Chair, ANPA Programs Committee
    Executive Director, Asniya, Inc.
    MRC 333, McLean Hospital
    Harvard Medical School
    115 Mill Street
    Belmont, MA  0²478
    office:     617.855.²051
    fax:         617.855.3199
    page:      617.855.²000

    Reply
    Posted by: Anonymous
    October 24, 2008 - 5:18 PM
  3. Thanks for this very helpful video. I'm interested in knowing where you purchased the freezing rod and ring that is used to form the sample mold. Alternatively what metal is the rod made of and is there any significance to the length of the freezing rod used?

    Reply
    Posted by: Ellen Y.
    April 23, 2010 - 3:57 PM

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