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Immunofluorescence Staining on Mouse Embryonic Brain Sections
小鼠胚胎脑切片的免疫荧光染色   

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Abstract

This protocol comprises the entire process of immunofluorescence staining on mouse embryonic brains, starting from tissue preparation to mounting of the tissue sections.

Keywords: Immunofluorescence staining(免疫荧光染色), Brain section(脑切片), Paraformaldehyde(多聚甲醛), Antibody(抗体), Cryostat(低温恒温器)

Materials and Reagents

  1. Paraformaldehyde (PFA)
    It can be ordered as 16% PFA from Electron Microscopy Sciences (catalog number: 15710 ), and diluted to 4% before use. However, we usually make the 4% solution from the powder (Sigma-Aldrich, catalog number: 158127 ). Here is how to make 4% PFA:
    1. In a 500 flask, weigh 4 g PFA in 80 ml PBS.
    2. Stir and heat the mixture on a magnetic heater. Keep the temperature below 60 °C during the entire process to avoid breakdown of the polymers.
    3. When temperature is close to 60 °C, add a few drops of 10 N sodium hydroxide. The mixture will gradually become clear.
    4. Filter the solution into a new flask and cool down to room temperature.
    5. Neutralize with hydrochloric acid to pH 7.
    6. Add PBS to 100 ml.
  2. O.C.T. (TFM Tissue Freezing Medium, TFM-5, clear color)
  3. Phosphate buffered saline (PBS)
  4. Goat serum
  5. Triton X-100
  6. Secondary antibodies:
    Cy2 (or cy3, or cy5)-conjugated goat-anti-mouse (or rabbit) IgG.
    (Jackson ImmunoResearch, F (ab) 2 fragments of affinity-purified antibody)
  7. Hoechst33258 (Sigma-Aldrich, catalog number: 861405 )
  8. Antifade reagent (Life Technologies, Invitrogen™, catalog number: P36930 )
  9. Ethanol
  10. 4 N hydrochloric acid
  11. Sodium hydroxide
  12. Blocking buffer (see Recipes)

Equipment

  1. Plastic cryomolds (Ted Pella, catalog number: 27110 )
  2. Cryostat sectioning machine
  3. SuperFrost Plus slides (VWR International, catalog number: 48311-703 )
  4. Dark humidified chamber: Made from a square petri dish containing kimwipes that are soaked in water. Wrap the chamber with aluminum foil to protect the slides from light.
  5. PAP pen
  6. 500 flask
  7. Ice bucket
  8. Magnetic heater

Procedure

  1. Perfuse embryos (E15-19) with 4% paraformaldehyde. Dissect out the brains and postfix in 4% paraformaldehyde overnight.
  2. In plastic cryomolds, embed brains in O.C.T., with the olfactory bulb facing downward.
  3. In an ice bucket, carefully drop some dry ice into 100 ml ethanol. When the bubbles stop popping, carefully transfer the mold into the dry ice-cooled ethanol. Watch the orientation of the brain before the O.C.T. freezes. Avoid splashing of ethanol into the mold. When the O.C.T. blocks completely freeze, store the block in -20 °C. The tissues are OK at -20 °C for two months, but best staining results are obtained on freshly frozen tissues.
  4. Section brains in cryostat sectioning machine, 10-20 μm thick. Attach brains to SuperFrost Plus slides. Air dry the sections for 20 min. The sections can be stored at -20 °C for two months, but best staining results are obtained on freshly cut tissues. Perform the following staining procedure in a dark humidified chamber.
  5. Wash with PBS, 3 times to clear out of the O.C.T. (Optional: For BrdU staining, brain sections were incubated in 4 N hydrochloric acid for 2 h at room temperature (RT) to unmask the antigen, followed by three washes in PBS).
  6. Slightly dry the slides, but make sure the tissue sections are kept wet. Use PAP pen to circle the four edges of the slides so that the staining solutions are confined in the circle.
  7. Add blocking buffer to the slides. Make sure that all tissue sections are covered with blocking buffer. Incubate at RT, 1 h.
  8. Aspirate blocking buffer, add primary antibody diluted in blocking buffer. Incubate at RT, 1 h (Or 4 °C, overnight). If more than one antibody is used in the staining, can add them simultaneously.
  9. Wash with PBS, 3 times, 5 min each.
  10. Add 2nd antibody diluted in blocking buffer, Incubate at RT, 1 h. If more than one antibody are used in the staining, can add the 2nd antibodies simultaneously. Don’t overstain with 2nd antibody!!
  11. Wash with PBS, 3 times, 5 min each.
  12. Stain the nuclei with Hoechst33258 (final concentration 1 μg/ml in PBS), at RT, 10 min.
  13. Wash with PBS, 3 times, 5 min each.
  14. Mount in antifade reagent.

Recipes

  1. Blocking buffer
    0.2% triton X-100 in PBS
    2% goat serum (The blocking serum should be from the same animal where the second antibody is produce)

Acknowledgments

This protocol was adapted from and used in Ge et al. (2009) and Mao et al. (2010).

References

  1. Ge, X., Frank, C. L., Calderon de Anda, F. and Tsai, L. H. (2010). Hook3 interacts with PCM1 to regulate pericentriolar material assembly and the timing of neurogenesis. Neuron 65(2): 191-203.
  2. Mao, Y., Ge, X., Frank, C. L., Madison, J. M., Koehler, A. N., Doud, M. K., Tassa, C., Berry, E. M., Soda, T., Singh, K. K., Biechele, T., Petryshen, T. L., Moon, R. T., Haggarty, S. J. and Tsai, L. H. (2009). Disrupted in schizophrenia 1 regulates neuronal progenitor proliferation via modulation of GSK3beta/beta-catenin signaling. Cell 136(6): 1017-1031.

简介

本协议旨在介绍通过经心灌注和提取大脑牺牲大鼠的方法,并介绍用c-Fos和Arc抗体染色大鼠脑组织的方法。 请注意,蛋白质的表达对触发神经活动的行为范例非常敏感。...

关键字:免疫荧光染色, 脑切片, 多聚甲醛, 抗体, 低温恒温器

材料和试剂

  1. 多聚甲醛(PFA)
    它可以作为16%PFA从Electron Microscopy Sciences(目录号:15710)订购,并在使用前稀释至4%。 然而,我们通常从粉末(Sigma-Aldrich,目录号:158127)制备4%的溶液。 这里是如何使4%PFA:
    1. 在500瓶中,称重80g PBS中的4g PFA
    2. 在磁力加热器上搅拌并加热混合物。 在整个过程中保持温度低于60°C,以避免聚合物的分解。
    3. 当温度接近60℃时,加入几滴10N氢氧化钠。 混合物将逐渐变清。
    4. 将溶液过滤到新的烧瓶中并冷却至室温。
    5. 用盐酸中和至pH为7.
    6. 加入PBS至100 ml。
  2. O.C.T. (TFM组织冷冻培养基,TFM-5,透明颜色)
  3. 磷酸盐缓冲盐水(PBS)
  4. 山羊血清
  5. Triton X-100
  6. 次级抗体:
    Cy2(或cy3或cy5)结合的山羊抗小鼠(或兔)IgG。
    (Jackson ImmunoResearch,经亲和纯化的抗体的F(ab)2片段)
  7. Hoechst33258(Sigma-Aldrich,目录号:861405)
  8. 抗褪色试剂(Life Technologies,Invitrogen TM,目录号:P36930)
  9. 乙醇
  10. 4 N盐酸
  11. 氢氧化钠
  12. 阻止缓冲区(参见配方)

设备

  1. 塑料低温模具(Ted Pella,目录号:27110)
  2. 冷冻切片机
  3. SuperFrost Plus载玻片(VWR International,目录号:48311-703)
  4. 黑暗加湿室:从包含浸泡在水中的kimwipes的方形培养皿制成。 用铝箔包裹室以保护载玻片免受光照。
  5. PAP笔
  6. 500瓶
  7. 冰桶
  8. 磁性加热器

程序

  1. 灌注胚胎(E15-19)与4%多聚甲醛。在4%多聚甲醛中解剖大脑和后缀一夜。
  2. 在塑料低温模型中,将大脑嵌入O.C.T.中,嗅球朝下。
  3. 在冰桶中,小心地将一些干冰滴入100ml乙醇中。当气泡停止爆裂时,小心地将模具转移到干冰冷却的乙醇中。在O.C.T.之前观察大脑的方向。冻结。避免乙醇溅入模具。当O.C.T.块完全冻结,将块储存在-20°C。组织在-20℃下可以保持两个月,但是在新鲜冷冻的组织上获得最好的染色结果
  4. 切片机的截面脑,厚度为10-20微米。将大脑连接到SuperFrost Plus幻灯片。空气干燥切片20分钟。切片可以在-20℃下储存两个月,但是在新鲜切割的组织上获得最好的染色结果。在黑暗的加湿室中进行以下染色程序
  5. 用PBS洗涤3次,以清除O.C.T. (可选:对于BrdU染色,将脑切片在室温(RT)下在4N盐酸中孵育2小时以暴露抗原,然后是三 在PBS中洗涤)
  6. 稍微干燥载片,但确保组织切片保持湿润。 使用PAP笔围绕载玻片的四个边缘,使染色溶液限制在圆形。
  7. 向幻灯片中添加阻塞缓冲区。 确保所有组织切片用封闭缓冲液覆盖。 在室温下孵育1小时。
  8. 吸出封闭缓冲液,加入在封闭缓冲液中稀释的一级抗体。 在室温下孵育1小时(或4℃,过夜)。 如果在染色中使用多于一种抗体,可以同时添加
  9. 用PBS清洗,3次,每次5分钟
  10. 加入在封闭缓冲液中稀释的2μL抗体,室温孵育1小时。 如果在染色中使用多于一种抗体,可以同时添加2种< sup>抗体。 不要用2 nd 抗体过度曝光!
  11. 用PBS洗涤,3次,每次5分钟。
  12. 用Hoechst33258(最终浓度为1μg/ml,在PBS中)染色细胞核,在室温下10分钟。
  13. 用PBS清洗,3次,每次5分钟
  14. 安装在防褪色剂中。

食谱

  1. 阻塞缓冲区
    0.2%triton X-100的PBS溶液 2%山羊血清(阻断血清应来自产生第二抗体的相同动物)

致谢

该协议改编自Ge等人(2009)和Mao等人(2010)中使用。

参考文献

  1. Ge,X.,Frank,C.L.,Calderon de Anda,F.and Tsai,L.H。(2010)。 Hook3与PCM1相互作用以调节近中心材料装配和神经发生的时间。 Neuron 65(2):191-203。
  2. Mao,Y.,Ge,X.,Frank,CL,Madison,JM,Koehler,AN,Doud,MK,Tassa,C.,Berry,EM,Soda,T.,Singh,KK,Biechele, ,TL,Moon,RT,Haggarty,SJ和Tsai,LH(2009)。 Cell 136(6):1017-1031。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ge, X. (2012). Immunofluorescence Staining on Mouse Embryonic Brain Sections. Bio-protocol 2(11): e192. DOI: 10.21769/BioProtoc.192.
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