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X-gal Staining on Adult Mouse Brain Sections

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The Journal of Neuroscience
May 2013


Knowing expression patterns of given proteins is very important to understand their functions. Immunostaining analysis with specific antibodies is commonly used to identify cells or tissues expressing proteins of interest. Although this technique is regularly used, it requires high quality of specific antibodies and there is no good quality of antibody available for certain proteins. Alternatively, X-gal staining is also used to analyze protein expression pattern. It is simple and routinely used to detect expression pattern of any proteins of interest in vivo. In this method, genetically modified animals that express beta-galactosidase under the control of certain regulatory elements will be used to reveal the expression pattern of proteins that use the same regulatory elements.

Keywords: Adult mouse brain (成年小鼠脑), X-gal staining (X-gal染色), Expression (表达)

Materials and Reagents

  1. Adult mouse brain
  2. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )
  3. Phosphate buffered saline (PBS)
  4. Sucrose
  5. Magnesium chloride, 6-Hydrate (MgCl2.6H2O) (Mallinckrodt Baker, catalog number: 2444-01 )
  6. Sodium deoxycholate (Sigma-Aldrich, catalog number: D6750 )
  7. NP-40 (Sigma-Aldrich, catalog number: I3021 )
  8. Potassium Ferricyanide (ACROS ORGANICS, catalog number: 196785000 )
  9. Potassium Ferrocyanide (Mallinckrodt Baker, catalog number: 6932-04 )
  10. Permount (Fisher Scientific, catalog number: SP15-100 )
  11. O.C.T. compund (Sakura, catalog number: 4583 )
  12. Ethanol (Decon Labs, catalog number: 2716 )
  13. X-gal (American Bioanatycal, catalog number: AB00450-00005 )
  14. 1 M MgCl2 (see Recipes)
  15. 10% Sodium deoxycholate (see Recipes)
  16. 20% NP-40 (see Recipes)
  17. 50 mM Potassium Ferricyanide (see Recipes)
  18. 50 mM Potassium Ferrocyanide (see Recipes)
  19. Staining buffer (see Recipes)


  1. Cryomold (Sakura, catalog number: 4557 )
  2. Cryostat
  3. 37 °C incubator
  4. 10-20 Slide Staining Dish with Cover (VWR International, catalog number: 900203 ) or equivalent


  1. Mouse is perfused with PBS for 5 min followed by 4% PFA/PBS for another 5 min.
  2. Mouse brain is dissected out and post-fixed with 4% PFA/PBS for 4 h at 4 °C.
  3. Fixed brain is washed with PBS three times and then incubated with 20% sucrose overnight (or until samples sink) and then 30% sucrose overnight.
  4. The brain is mounted on standard cryomold with O.C.T. compound and stored at -80 °C until usage.
  5. 40 μm cyro-section is made using cryostat and mounted on the slide-glass.
  6. Slide-glass is washed once with the staining buffer (typically 150 ml for a container) for 10 min at room temperature.
  7. Slide-glass is then incubated with 1 mg/ml X-gal in the staining buffer supplemented with 5 mM Potassium Ferricyanide and 5 mM Potassium Ferrocyanide at 37 °C until color develops.
    1. This step usually takes 3 h to overnight.
    2. Don’t let samples dry.
    3. Use 100-200 μl X-gal solutions per slide.
  8. Stained sample is washed with PBS three times (5 min each).
  9. Slide is dehydrated with series of ethanol (50%, 75%, 90%, 100%, 2 min each).
  10. Slide is mounted on permount and can be stored at room temperature.
  11. Sample is ready for imaging at higher magnification (Figure 1).

    Figure 1. X-gal staining of Nlk gene trap mouse brain. Strong beta-galactosidase expression of the Nlk gene trap mouse is observed within the Purkinje cell layer from 6-week-old Nlk gene trap mouse cerebellum.  MCL, molecular cell layer.  PCL, Purkinje cell layer.  GCL, granular cell layer.


  1. The duration of incubation depends on how strongly beta-galactosidase is expressed. Sometimes it requires longer than 2 days of incubation. You can use fresh X-gal solution after overnight incubation.
  2. Wet tissue must be put in the staining container.


  1. 1 M MgCl2
    Add 10.17 g of MgCl2 into 40 ml dH2O
    Add dH2O to 50 ml
    Stored at room temperature
  2. 10% Sodium deoxycholate
    Add 5 g of Sodium deoxycholate into 40 ml dH2O
    Add dH2O to 50 ml
    Stored at room temperature
  3. 20% NP-40
    Mix 10 ml NP-40 with 40 ml dH2O
    Stored at room temperature
  4. 50 mM Potassium Ferricyanide
    Add 16.46 g of Potassium Ferricyanide into 800 ml dH2O
    Add dH2O to 1,000 ml
    Stored at 4 °C
    Protect from light
  5. 50 mM Potassium Ferrocyanide
    Add 21.12 g of Potassium Ferricyanide into 800 ml dH2O
    Add dH2O to 1,000 ml
    Stored at 4 °C
    Protect from light
  6. Staining buffer
    Mix 2 ml of 1 M MgCl2, 1 ml of 10% sodium deocycholate, 1 ml of 20% NP-40 with 800 ml dH2O
    Add dH2O to 1,000 ml
    Stored at room temperature


The authors would like to thank the members of the Lim Lab for feedback on this manuscript. This protocol was adapted from the previously published paper: Ju et al. (2013). This work was supported by the National Institute of Neurological Disorders and Stroke grant NS064146, the Brain and Behavior Research Foundation (Formerly NARSAD), the Alfred P. Sloan Foundation, the National Multiple Sclerosis Society, the Charles H. Hood Foundation, the National Ataxia Foundation, and the Yale Scholar Award Program to J. Lim.


  1. Ju, H., Kokubu, H., Todd, T. W., Kahle, J. J., Kim, S., Richman, R., Chirala, K., Orr, H. T., Zoghbi, H. Y. and Lim, J. (2013). Polyglutamine disease toxicity is regulated by Nemo-like kinase in spinocerebellar ataxia type 1. J Neurosci 33(22): 9328-9336.


了解给定蛋白质的表达模式对于了解其功能非常重要。 特异性抗体的免疫染色分析通常用于鉴定表达目的蛋白质的细胞或组织。 虽然这种技术是经常使用的,但它需要高质量的特异性抗体,并且对某些蛋白质没有可用的抗体质量很好。 或者,X-gal染色也用于分析蛋白质表达模式。 简单且常规地用于检测任何目标蛋白在体内的表达模式。 在这种方法中,在某些调节元件控制下表达β-半乳糖苷酶的转基因动物将用于揭示使用相同调控元件的蛋白质的表达模式。

关键字:成年小鼠脑, X-gal染色, 表达


  1. 成年老鼠大脑
  2. 多聚甲醛(PFA)(Sigma-Aldrich,目录号:P6148)
  3. 磷酸盐缓冲盐水(PBS)
  4. 蔗糖
  5. 氯化镁,6-水合物(MgCl 2·6H 2 O·6H 2 O)(Mallinckrodt Baker,目录号:2444-01)
  6. 脱氧胆酸钠(Sigma-Aldrich,目录号:D6750)
  7. NP-40(Sigma-Aldrich,目录号:I3021)
  8. 铁氰化钾(ACROS ORGANICS,目录号:196785000)
  9. 氰亚铁酸钾(Mallinckrodt Baker,目录号:6932-04)
  10. Permount(Fisher Scientific,目录号:SP15-100)
  11. O.C.T. compund(Sakura,目录号:4583)
  12. 乙醇(Decon Labs,目录号:2716)
  13. X-gal(American Bioanatycal,目录号:AB00450-00005)
  14. 1 M MgCl <2> (参见配方)
  15. 10%脱氧胆酸钠(参见配方)
  16. 20%NP-40(见配方)
  17. 50 mM铁氰化钾(见配方)
  18. 50 mM亚铁氰化钾(见配方)
  19. 染色缓冲液(见配方)


  1. Cryomold(Sakura,目录号:4557)
  2. 冷冻切片机
  3. 37℃孵育器
  4. 10-20带盖的幻灯片污渍盘(VWR International,目录号:900203)或等同物


  1. 小鼠用PBS灌注5分钟,然后用4%PFA/PBS再灌注5分钟
  2. 解剖出小鼠脑,并在4℃下用4%PFA/PBS后固定4小时
  3. 固定的脑用PBS洗涤三次,然后用20%蔗糖孵育过夜(或直到样品下沉),然后用30%蔗糖过夜。
  4. 将大脑安装在具有O.C.T的标准低温模型上。 化合物并储存在-80℃直至使用
  5. 40微米的截面是使用低温恒温器并安装在载玻片上
  6. 载玻片用染色缓冲液(通常为150ml的容器)在室温下洗涤10分钟
  7. 然后将载玻片在补充有5mM铁氰化钾和5mM氰亚铁酸钾的染色缓冲液中在37℃下与1mg/ml X-gal一起孵育,直到颜色发展。
    1. 此步骤通常需要3小时至隔夜。
    2. 不要让样品变干。
    3. 每张幻灯片使用100-200μlX-gal溶液。
  8. 用PBS洗涤染色的样品三次(每次5分钟)
  9. 载玻片用一系列乙醇(50%,75%,90%,100%,每次2分钟)脱水
  10. 载玻片安装在固定架上,可在室温下储存。
  11. 样品准备好以更高的放大倍数成像(图1)

    图1.Nlk 基因捕获小鼠脑的X-gal染色。在Nlk 基因捕获小鼠中观察到强的β-半乳糖苷酶表达 来自6周龄的Nlk 基因捕获小鼠小脑的Purkinje细胞层。 MCL,分子细胞层。 PCL,浦肯野细胞层。 GCL,颗粒细胞层。


  1. 孵育的持续时间取决于β-半乳糖苷酶的表达有多强。 有时它需要超过2天的孵化。 您可以在过夜孵育后使用新鲜的X-gal溶液
  2. 湿织物必须放在染色容器中。


  1. 1 M MgCl 2
    将10.17g MgCl 2加入到40ml dH 2 O中。
    将dH <2> O添加到50ml
  2. 10%脱氧胆酸钠 将5g脱氧胆酸钠加入到40ml dH 2 O中 将dH <2> O添加到50ml
  3. 20%NP-40
    将10ml NP-40与40ml dH 2 O混合 在室温下贮存
  4. 50 mM铁氰化钾
    将16.46g铁氰化钾加入到800ml dH 2 O中 将dH <2> O添加至1,000 ml
  5. 50 mM氰亚铁酸钾
    将21.12g铁氰化钾加入到800ml dH 2 O中 将dH <2> O添加至1,000 ml
  6. 染色缓冲区
    将2ml 1M MgCl 2,1ml 10%脱氢胆酸钠,1ml 20%NP-40与800ml dH 2 O混合。 将dH <2> O添加至1,000 ml


作者要感谢林实验室的成员对这份手稿的反馈。 该协议改编自以前发表的论文:Ju em et al。(2013)。 这项工作得到国家神经疾病和中风研究所NS064146,大脑和行为研究基金会(原NARSAD),阿尔弗雷德·斯隆基金会,国家多发性硬化症协会,查尔斯·Hood基金会,国家共济失调 基金会和耶鲁学者奖计划。


  1. Ju,H.,Kokubu,H.,Todd,T.W.,Kahle,J.J.,Kim,S.,Richman,R.,Chirala,K.,Orr,H.T.,Zoghbi,H.Y.and Lim, 多谷氨酰胺疾病的毒性是由1型脊髓小脑性共济失调中的Nemo样激酶调节的。 em> J Neurosci 33(22):9328-9336。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Kokubu, H. and Lim, J. (2014). X-gal Staining on Adult Mouse Brain Sections. Bio-protocol 4(5): e1064. DOI: 10.21769/BioProtoc.1064.
  2. Ju, H., Kokubu, H., Todd, T. W., Kahle, J. J., Kim, S., Richman, R., Chirala, K., Orr, H. T., Zoghbi, H. Y. and Lim, J. (2013). Polyglutamine disease toxicity is regulated by Nemo-like kinase in spinocerebellar ataxia type 1. J Neurosci 33(22): 9328-9336.