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Fluoro-Jade B Staining for Neuronal Cell Death
神经元细胞死亡的Fluoro-Jade B染色   

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Abstract

Fluoro-Jade is a fluorescent derivative used for histological staining of degenerating neurons. This technique is simple and sensitive enough to label distal dendrites, axons, axon terminals as well as neuronal bodies. Fluoro-Jade has excitation and emission peak of 480 and 525 nanometer respectively. It can be visualized using a fluorescein/FITC filter. Some reports have demonstrated that Fluoro-Jade can also be useful to detect glial cell death (Anderson et al., 2013; Damjanac et al., 2007).

Keywords: Fluoro-Jade B(Fluoro-JadeB), Brain staining(脑染色), Cell death(细胞死亡), Neurone(神经元)

Materials and Reagents

  1. Superfrost plus Microscope slide (Thermo Fisher Scientific, catalog number: 12-550-17 )
  2. Cover Glass (Thermo Fisher Scientific, catalog number: 12-545-88 )
  3. Tissue sample
  4. Fluoro-Jade B (Merck Millipore Corporation, catalog number: AG310 )
  5. Paraformaldehyde (Electron Microscopy Science, catalog number: 19210 )
  6. Potassium permanganate (KMnO4) (Sigma-Aldrich, catalog number: 223468 )
  7. DAPI (Life Technologies, catalog number: D3571 )
    Note: Currently, it is “Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D3571”.
  8. Glacial Acetic acid (CH3CO2H) (Sigma-Aldrich, catalog number: A9967 )
  9. Ethanol
  10. Xylene (Sigma-Aldrich, catalog number: 534056 )
  11. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S8045 )
  12. Sodium tetraborate decahydrate (Sigma-Aldrich, catalogue number: B9876 )
  13. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
  14. potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: P3786 )
  15. potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P9791 )
  16. 4% paraformaldehyde (see Recipes)
  17. KPBS (see Recipes)
  18. 0.2% Fluoro-Jade (see Recipes)
  19. Fluoro-Jade solution (see Recipes)
  20. 0.2% DAPI (see Recipes)

Equipment

  1. Vacuum Desiccators (Thermo Fisher Scientific, catalog number: 08-642-5 )
  2. Tissue tek slide staining set (Electron Microscopy Science, catalog number: 62540-01 )
  3. 24 slide holder (Electron Microscopy Science, catalog number: 62543-06 )
  4. Orbital shaker
  5. Timer
  6. Slide Warmer
  7. DPX mounting medium (a mixture of the polystyrene distyrene and the plasticizer dibutylphthalate) (Electron Microscopy Science, catalog number: 13512 )

Procedure

  1. Mount tissue sections (20-35 μm cut on microtome or cryostat) on superfrost plus slide and let the slide dry overnight under vacuum.
  2. Fix the tissue on slide warmer 30 min at 60 °C and/or 20 min in paraformaldehyde 4%.
  3. Follow by 2 min in KPBS.
    Note: All the following steps are done at room temperature.
  4. Dehydrate in 50%-70%-100% Ethanol 2 min each.
  5. Rehydrate by going back in 70%-50% Ethanol and KPBS, 2 min each.
  6. Incubate in potassium permanganate 0.06% (dilute in water) 5 min at room temperature.
  7. Rinse in water 1 min.
  8. Incubate in Fluoro-Jade solution at room temperature for 10 min and gently shake on orbital shaker or by doing several dips during incubation (three dips of few seconds 3 times during incubation). Use opaque cup for the Fluoro-Jade incubation and keep the slide in shelters from light for the rest of the procedure.
    Note: Fluoro-Jade solution and potassium permanganate 0.06% must be prepared fresh.
  9. Follow by three rinses of 1 min each in water.
  10. Dry the slides overnight under vacuum at room temperature.
  11. Clear the slide in Xylene (3 x 2 min).
  12. Cover slip with DPX and dry 24-48 h under hood before microscope analysis.


    Figure 1. Example of the set up use for incubation and/or dipping of the slides in different solutions

Representative data


Figure 2. FJB-positive neurons in the mouse cerebral cortex following ischemic stroke

Notes

To ensure reproducibility between protocols, use the same method of tissue preparation.

Recipes

  1. 4% paraformaldehyde
    Heat 700 ml distilled water at 65 °C
    Add 40 g paraformaldehyde
    5 ml NaOH
    When Paraformaldehyde is completely dissolved add 38 g sodium tetraborate
    Complete at 1 liter with distilled water
  2. KPBS
    3.81 g Potassium phosphate dibasic
    0.45 g Potassium phosphate monobasic
    8.1 g sodium chloride
    Complete to 1 liter with distilled water
  3. 0.2% Fluoro-Jade
    Dilute 50 mg Fluoro-Jade B in 25 ml of sterile water and aliquot
    Keep this stock solution at -20 °C in shelters from light
  4. Fluoro-Jade solution
    Fluoro-Jade B 0.0004%
    Acetic acid 0.1%
    DAPI 0.0001% in water
  5. 0.2% DAPI
    Dilute 10 mg DAPI in 5 ml of sterile water and aliquot
    Keep this stock solution at 4 °C in shelters from light

Acknowledgments

This protocol was adapted from Schmued et al. (1997). This work was supported by grants from the Canadian Institutes for Health Research (CIHR) and the Multiple Sclerosis Scientific Research Foundation of Canada.

References

  1. Anderson, K. J., Fugaccia, I. and Scheff, S. W. (2003). Fluoro-Jade B stains quiescent and reactive astrocytes in the rodent spinal cord. J Neurotrauma 20(11): 1223-1231.
  2. Damjanac, M., Rioux Bilan, A., Barrier, L., Pontcharraud, R., Anne, C., Hugon, J. and Page, G. (2007). Fluoro-Jade B staining as useful tool to identify activated microglia and astrocytes in a mouse transgenic model of Alzheimer's disease. Brain Res 1128(1): 40-49.
  3. Schmued, L. C., Albertson, C. and Slikker, W., Jr. (1997). Fluoro-Jade: a novel fluorochrome for the sensitive and reliable histochemical localization of neuronal degeneration. Brain Res 751(1): 37-46.

简介

Fluoro-Jade是用于退化神经元的组织学染色的荧光衍生物。 这种技术是简单和足够敏感到标记远端树突,轴突,轴突终端以及神经元体。 翡翠的激发和发射峰分别为480和525纳米。 它可以使用荧光素/FITC过滤器可视化。 一些报道已经证明氟 - 玉也可以用于检测胶质细胞死亡(Anderson等人,2013; Damjanac等人,2007)。

关键字:Fluoro-JadeB, 脑染色, 细胞死亡, 神经元

材料和试剂

  1. Superfrost plus显微镜载玻片(Thermo Fisher Scientific,目录号:12-550-17)
  2. 盖玻璃(Thermo Fisher Scientific,目录号:12-545-88)
  3. 组织样品
  4. Fluor-Jade B(Merck Millipore Corporation,目录号:AG310)
  5. 多聚甲醛(Electron Microscopy Science,目录号:19210)
  6. 高锰酸钾(KMnO 4)(Sigma-Aldrich,目录号:223468)
  7. DAPI(Life Technologies,目录号:D3571)
    注意:目前,"Thermo Fisher Scientific,Molecular Probes TM ,目录号:D3571"。
  8. 冰乙酸(CH 3 CO 2 H)(Sigma-Aldrich,目录号:A9967)
  9. 乙醇
  10. 二甲苯(Sigma-Aldrich,目录号:534056)
  11. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:S8045)
  12. 十水合四硼酸钠(Sigma-Aldrich,目录号:B9876)
  13. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S3014)
  14. 磷酸二氢钾(K 2 HPO 4)(Sigma-Aldrich,目录号:P3786)
  15. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P9791)
  16. 4%多聚甲醛(见配方)
  17. KPBS(请参阅配方)
  18. 0.2%氟玉(见配方)
  19. Fluoro-Jade解决方案(参见配方)
  20. 0.2%DAPI(参见配方)

设备

  1. 真空干燥器(Thermo Fisher Scientific,目录号:08-642-5)
  2. 组织片幻灯片染色组(Electron Microscopy Science,目录号:62540-01)
  3. 24载片架(Electron Microscopy Science,目录号:62543-06)
  4. 轨道振动器
  5. 计时器
  6. 幻灯片加温器
  7. DPX安装介质(聚苯乙烯二苯乙烯和增塑剂邻苯二甲酸二丁酯的混合物)(Electron Microscopy Science,目录号:13512)

程序

  1. 在superfrost plus载玻片上安装组织切片(在切片机或低温恒温器上切割20-35μm),让载玻片在真空下干燥过夜。
  2. 固定组织在幻灯片加热30分钟在60℃和/或20分钟在多聚甲醛4%
  3. 在KPBS后2分钟。
    注意:以下所有步骤都是在室温下进行。
  4. 在50%-70%-100%乙醇中脱水2分钟。
  5. 通过回到70%-50%乙醇和KPBS中再水合,每次2分钟。
  6. 在室温下在高锰酸钾中孵育0.06%(在水中稀释)5分钟。
  7. 在水中冲洗1分钟。
  8. 在氟 - 玉溶液中在室温下孵育10分钟并在定轨振荡器上轻轻摇动或通过在孵育期间进行几次浸渍(孵育期间三次浸泡几秒钟3次)。使用不透明的杯子为氟玉孵化,并保持幻灯片在避难所从光为其余的程序。
    注意:氟玉溶液和高锰酸钾0.06%必须新鲜制备。
  9. 随后在水中漂洗三次,每次1分钟
  10. 在室温下在真空下干燥载玻片过夜
  11. 清除二甲苯中的幻灯片(3 x 2分钟)。
  12. 在显微镜分析前,用DPX盖住玻片并在罩下干燥24-48小时

    图1.用于孵化和/或在不同解决方案中浸渍幻灯片的设置示例

代表数据


图2.缺血性中风后小鼠大脑皮质中的FJB阳性神经元

笔记

为了确保方案之间的重现性,使用相同的组织制备方法。

食谱

  1. 4%多聚甲醛
    在65℃下加热700ml蒸馏水
    加入40克多聚甲醛
    5 ml NaOH
    当多聚甲醛完全溶解时,加入38g四硼酸钠
    用蒸馏水以1升完成。
  2. KPBS
    3.81g磷酸氢二钾
    0.45克磷酸二氢钾
    8.1克氯化钠
    用蒸馏水
    完成至1升
  3. 0.2%氟玉
    在25ml无菌水中稀释50mg氟 - 玉B,并等分
    将此储备液在-20℃避光保存,避光
  4. 氟玉溶液
    Fluoro-Jade B 0.0004%
    乙酸0.1%
    DAPI 0.0001%水溶液
  5. 0.2%DAPI
    稀释10mg DAPI在5ml无菌水中,并等分
    将此储备溶液保持在4°C,避光避光

致谢

该协议改编自Schmued等人(1997)。这项工作得到加拿大健康研究所(CIHR)和加拿大多发性硬化症科学研究基金会的资助。

参考文献

  1. Anderson,K.J.,Fugaccia,I.and Scheff,S.W。(2003)。 Fluoro-Jade B在啮齿动物脊髓中染色静止和反应性星形胶质细胞。 J Neurotrauma 20(11):1223-1231。
  2. Damjanac,M.,Rioux Bilan,A.,Barrier,L.,Pontcharraud,R.,Anne,C.,Hugon,J。和Page,G。 Fluoro-Jade B染色作为在阿尔茨海默病小鼠转基因模型中鉴定活化的小胶质细胞和星形胶质细胞的有用工具疾病。 Brain Res 1128(1):40-49
  3. Schmued,L.C.,Albertson,C。和Slikker,W.,Jr。(1997)。 Fluoro-Jade:一种用于敏感和可靠的神经元变性的组织化学定位的新颖荧光染料。 Brain Res 751(1):37-46。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Laflamme, N., Préfontaine, P. and Rivest, S. (2016). Fluoro-Jade B Staining for Neuronal Cell Death. Bio-protocol 6(1): e1702. DOI: 10.21769/BioProtoc.1702.
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