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

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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

神经科学 > 发育 > 免疫荧光
作者: Xuecai Ge
Xuecai GeAffiliation 1: Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology (MIT), Cambridge, USA
Affiliation 2: , Howard Hughes Medical Institute, Cambridge, USA
For correspondence: xuecaige@stanford.edu
Bio-protocol author page: a46
Vol 2, Iss 11, 6/5/2012, 11073 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.192

[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)

[Abstract] 这个实验方法包括小鼠胚胎脑切片的免疫荧光染色的整个过程,从组织制备到封片。

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.

材料和试剂

 

1.        低聚甲醛 (PFA)

16% PFA 可以从公司订购,使用前再将其稀释到4%。我通常用粉末(Sigma 158127)配制4% 的溶液,下面是配置方法:

1)      在一个 500瓶子里,称取4g PFA放入80ml PBS中。

2)      用磁性加热搅拌器搅拌混合液体。在整个过程中保持温度在60度以下,防止聚合物解聚。

3)      当温度接近60oC, 加几滴10N 氢氧化钠. 混合物将逐渐的变清澈。

4)      在新瓶子中过滤溶液,冷却到室温。

5)      用盐酸中和到PH 7.

6)      PBS  100ml.

2.        O.C.T. (TFM 组织冷冻 Medium, TFM-5, 澄清)

3.        PBS

4.        山羊血清

5.        Triton X-100

6.        二抗:

Cy2 (or Cy3, or Cy5)-接合 羊抗鼠 (or 兔子) IgG. (Jackson ImmunoResearch, F(ab)2 亲和纯化抗体。)

7.        Hoechst33258 (Sigma 861405)

8.        抗衰退试剂 (Invitrogen P36930)

 

设备

 

1.        塑料cryomolds (TedPella, #27110)

2.        冷冻切片机

3.        SuperFrost Plus 载玻片 (VWR 48311-703)

4.        避光增湿箱: 用正方形培养皿制作,包含湿巾在水中浸泡,用铝箔纸包上防止切片曝光。

5.        PAP pen

 

步骤

 

1.        4%低聚甲醛灌满胚胎 (E15-19).切开脑组织用 4%低聚甲醛过夜固定。

2.        用塑料cryomolds,O.C.T包埋脑组织, 加上一串嗅球。

3.        在一个冰盒中,小心的滴入干冰到100ml乙醇中,当停止冒泡,小心的将包埋体加入其中。当 O.C.T.冻结前观察脑组织的方向。当O.C.T.完全冻结时,防止乙醇进入腔室,将包埋体存入 -20oC,可以保存2个月。但是最好新鲜的冷度组织染色,这样得到的结果会更好。

4.        用冷冻切片机切片到10-20um厚,将脑组织放到 SuperFrost Plus载玻片上,空气干燥切片20min,可以在-20oC 保存两个月,但是最好用新鲜的冷冻组织染色。做下面步骤实验时,要在有湿度的暗箱中进行。

5.         PBS3 次,清除O.C.T. (可选择: 对于BrdU 染色,脑切片在4N 的盐酸中室温下孵育两个小时,暴露抗体,然后再用PBS洗涤3)

6.        使载玻片稍微干燥一下,但是要确保组织切片组织保持湿润。使用PAP封闭四种,使得溶液在里面。

7.        加入封闭液到载玻片,确保所有的组织切片都在封闭缓冲液中,在室温下孵育1个小时。

8.        吸出封闭液,在封闭液中加入抗,在室温下孵育1个小时,或者四度过夜。如果需要加多种抗体染色,可以同时加。

9.        PBS3 times, 5min 一次.

10.    在封闭液中加二抗,在室温下孵育1个小时,如果需要加多种二抗,可以同时加。 不要用二抗过度染色!!

11.    5分钟一次,用PBS洗涤三次。

12.    Hoechst33258 染核(终浓度 1ug/ml ,用PBS稀释),在室温下 10min.

13.     PBS3,5min 每次.

14.    在抗衰剂 (Invitrogen P36930)中封藏。

 

配置方法

 

1.        封闭液:

0.2% triton X-100  PBS

2% 山羊血清 (封闭血清必须与二抗提供的动物为相同动物)

 

参考文献

 

1.         Ge X., Frank C.L., Calderon de Anda F., 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., Tsai L.H. (2009). Disrupted in schizophrenia 1 regulates neuronal progenitor proliferation via modulation of GSK3beta/beta-catenin signaling. Cell 136(6): 1017-31. 

 

 

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How to cite this protocol: Ge, X. (2012). Immunofluorescence Staining on Mouse Embryonic Brain Sections. Bio-protocol 2(11): e192. DOI: 10.21769/BioProtoc.192; Full Text



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