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TRIPLE (Insulin, Glucagon and EGFP) Immunofluorescence Staining Protocol in Pancreas
胰腺的三重(胰岛素、胰高血糖素和增强型绿色荧光蛋白(EGFP))免疫荧光染色法   

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Abstract

This protocol aims to introduce methods for immunostaining two endogenous proteins insulin and glucagon and one exogenous transgene driven EGFP in mouse pancreatic islet. The immunostaining results of insulin and glucagon indirectly tell functionality of pancreatic beta cells and alpha cells respectively. Furthermore, the protocol provides immunostaining steps for the third protein which can be applicable to any other endogenous proteins with a specific antibody generated in mouse.

Materials and Reagents

  1. Mouse pancreas
  2. Ethanol (100%, 95%, 85%, 70%, 50%)
  3. 0.85% NaCl
  4. ddH2O
  5. 99.5% Xylene (Sigma-Aldrich, catalog number: 534056 )
  6. 10% Formalin solution (neutral buffered) (Sigma-Aldrich, catalog number: HT501128 )
  7. Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A7906 )
  8. Tween 20 (Sigma-Aldrich, catalog number: P9416 )
  9. Triton-X 100 (Sigma-Aldrich, catalog number: T8787 )
  10. Guinea pig anti-insulin antibody (Millipore, catalog number: 4011-01F )
  11. Rabbit anti-glucagon antibody (Millipore, catalog number: 4030-01F )
  12. Mouse anti-EGFP antibody (Clonetech, catalog number: 632381 )
  13. Texas Red conjugated donkey anti-guinea pig antibody (Jackson ImmunoResearch Laboratories, catalog number: 106-075-003 )
  14. Alexa 350 conjugated goat anti-rabbit antibody (Life Technologies, InvitrogenTM, catalog number: A-21068 )
  15. FITC conjugated goat anti-mouse antibody (Jackson ImmunoResearch Laboratories, catalog number: 715-095-166 )
  16. ProLong Gold Antifade Reagent (Life Technologies, InvitrogenTM, catalog number: P36935 )
  17. 10x PBS (Gibco®, catalog number: 70011-044 )
  18. Blocking solution (see Recipes)

Equipment

  1. Glass jar
  2. Humidified chamber
  3. Confocal microscope
  4. Tissue cassette

Procedure

  1. Formalin Fixation
    1. Fix overnight pancreas (~5mm x ~5mm) removed from a mouse in a 15 ml conical tube by 10 ml 10% formalin solution during rotating it at 4 °C.
    2. On next day, transfer the fixed tissue to a tissue cassette and store it in 70% Ethanol solution till making paraffin block (for at least 1 day) at room temperature.
    3. Make paraffin blocks of fixed tissues.
    4. Make slide section of paraffin blocks by Microtome.

  2. Immunostaining of slide sections
    1. Deparaffinization step
      1. Keep sections for 5 min in 99.5% Xylene solution 2 times.
      2. Keep sections for 10 min in 99.5% Xylene solution 2 times.
      3. Keep sections for 3 min in 100% Ethanol with new glass jar.
      4. Keep sections for 3 min in 95% Ethanol.
      5. Keep sections for 3 min in 85% Ethanol.
      6. Keep sections for 3 min in 70% Ethanol.
      7. Keep sections for 5 min in 50% Ethanol.
      8. Keep sections for 5 min in 0.85% NaCl with new glass jar.
      9. Keep sections for 5 min in 1x PBS.
    2. Permeabilization step
      1. Incubate sections for 30 min in 0.1% Triton-X 100 in PBS with new glass jar at room temperature (RT).
      2. Incubate sections for 5 min in 1x PBS with new glass jar 3 times.
    3. Blocking, Primary and Secondary Antibody (Ab) binding steps
      1. Aspirate around the tissue until the slide, not the tissue, is dry. Carefully, trace around the tissue with a grease choke.
      2. Block for 30 min at RT with 2% BSA in PBS with 0.05% Tween 20 in a coplin jar.
      3. Aspirate blocking buffer and incubate sections in a humidified camber with glucagon primary Ab (1/100 dilution), in 2% BSA in PBS with 0.05% Tween 20 for overnight (o/n) at 4 °C. Use enough antibody solution (~150 μl) to completely submerge the section.
      4. Aspirate primary antibody and wash sections for 5 min in 2% BSA in PBS with 0.05% Tween 20 2 times in a coplin jar.
      5. Aspirate washing buffer and incubate sections in a humidified chamber with corresponding secondary Alexa350 conjugated goat anti-rabbit Ab (1/500 dilution) for 1.15 h at RT in dark. Dilute secondary Ab in 2% BSA in PBS with 0.05% Tween 20. Use enough antibody solution (~150 μl) to completely submerge the section.
      6. Aspirate secondary antibody and wash sections for 5 min in 2% BSA in PBS with 0.05% Tween 20 2 times in a coplin jar. Aspirate around the tissue until the slide, not the tissue, is dry. And incubate sections in a humidified chamber with Insulin primary Ab (1/500 dilution), in 2% BSA in PBS with 0.05% Tween 20 for 1.5 h at RT in dark. Use enough antibody solution (~150 μl) to completely submerge the section.
      7. Aspirate primary antibody and wash sections for 5 min in 2% BSA in PBS with 0.05% Tween 20 2 times at RT in dark in a coplin jar.
      8. Aspirate around the tissue until the slide, not the tissue, is dry. And Incubate sections in a humidified chamber with corresponding secondary Texas Red conjugated donkey anti-guinea pig Ab for 1 h at RT in dark. Dilute secondary Ab (1/1,000 dilution) in 2% BSA in PBS with 0.05% Tween 20. Use enough antibody solution (~150 μl) to completely submerge the section.
      9. Aspirate secondary antibody and wash sections for 5 min in 2% BSA in PBS with 0.05% Tween 20 2 times at RT in dark in a coplin jar. Aspirate around the tissue until the slide, not the tissue, is dry. And incubate sections in a humidified camber with EGFP primary Ab (1/500 dilution), for 1 h at 4 °C in dark. Diluted primary Ab in 2% BSA in PBS with 0.05% Tween 20. Use enough antibody solution (~150 μl) to completely submerge the section.
      10. Aspirate primary antibody and wash sections for 5 min in 2% BSA in PBS with 0.05% Tween 20 2 times at RT in dark in a coplin jar.
      11. Aspirate washing buffer and Incubate sections in a humidified chamber with corresponding secondary FITC conjugated goat anti-mouse Ab (1/500 dilution) for 1 h at RT in dark. Dilute secondary Ab in 2% BSA in PBS with 0.05% Tween 20. Use enough antibody solution (~150 μl) to completely submerge the section.
      12. Aspirate secondary antibody and wash sections for 5 min in 2% BSA in PBS with 0.05% Tween 20 2 times at RT in dark in a coplin jar.

  3. Mounting Step
    1. Aspirate around the tissue until the slide, not the tissue, is dry, Add ~50 μl of ProLong® Gold Antifade Reagent containing DAPI to each section and place a cover slip over section.
      Note: Be careful to avoid damaging the tissue by sliding the cover slip too much and do not introduce bubbles. Aspirate excess reagent.
    2. Dry sections at room temperature for several hours or overnight protected from light.
    3. Once the slides are completely dry, seal the edges of the cover slip with clear nail polish. Slides can be stored at -20 °C, protected from light for several weeks.

Recipes

  1. Blocking solution (2% BSA in PBS pH 7.4 with 0.05% Tween20)
    10 g BSA
    50 ml 10x PBS
    0.25 ml Tween20
    Add ddH2O to 500 ml

Representative Data



Figure 1. Triple immunostaining for EGFR, insulin, and glucagon in a pancreatic tissue section. Pancreatic tissue sections were obtained from mice 3 weeks after Tam administration to induce EGFP from a transgene. Pancreatic tissue sections were triple immunostained for EGFP (Green), insulin (Red), and glucagon (Blue), and representative single-channel fluorescence images are shown individually and merged (lower-right image of each group). [Please cite Reference 1 (Figure 1B)]

Acknowledgments

This work was supported in part by the National Research Foundation of Korea funded by the Ministry of Education, Science, and Technology Grants 2011-0011433, 2012M3A9C3048686 and 2014R1A1A4A01004329 (S.H.B) and NIH grants DK42394, HL52173, and HL057346 (R.J.K.). R.J.K. was an Investigator of the Howard Hughes Medical Institute. This protocol was adapted from Back et al. (2009), and a first short version of the adapted protocol was published in Han et al. (2013).

References

  1. Back, S. H.*, Scheuner, D.*, Han, J., Song, B., Ribick, M., Wang, J., Gildersleeve, R. D., Pennathur, S. and Kaufman, R. J. (2009). Translation attenuation through eIF2alpha phosphorylation prevents oxidative stress and maintains the differentiated state in beta cells. Cell Metab 10(1): 13-26. *These authors contributed equally to this work
  2. Han, J.*, Back, S. H.*, Hur, J., Lin, Y. H., Gildersleeve, R., Shan, J., Yuan, C. L., Krokowski, D., Wang, S., Hatzoglou, M., Kilberg, M. S., Sartor, M. A. and Kaufman, R. J. (2013). ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death. Nat Cell Biol 15(5): 481-490. *These authors contributed equally to this work

简介

该协议旨在介绍在小鼠胰岛中免疫染色两种内源性蛋白质胰岛素和胰高血糖素和一种外源性转基因驱动的EGFP的方法。 胰岛素和胰高血糖素的免疫染色结果分别间接地指示胰腺β细胞和α细胞的功能。 此外,该方案提供了第三种蛋白质的免疫染色步骤,其可以应用于任何其他内源性蛋白质与在小鼠中产生的特异性抗体。

材料和试剂

  1. 小鼠胰腺
  2. 乙醇(100%,95%,85%,70%,50%)
  3. 0.85%NaCl
  4. ddH sub 2 O
  5. 99.5%二甲苯(Sigma-Aldrich,目录号:534056)
  6. 10%福尔马林溶液(中性缓冲液)(Sigma-Aldrich,目录号:HT501128)
  7. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A7906)
  8. 吐温20(Sigma-Aldrich,目录号:P9416)
  9. Triton-X 100(Sigma-Aldrich,目录号:T8787)
  10. 豚鼠抗胰岛素抗体(Millipore,目录号:4011-01F)
  11. 兔抗胰高血糖素抗体(Millipore,目录号:4030-01F)
  12. 小鼠抗EGFP抗体(Clonetech,目录号:632381)
  13. 德克萨斯红缀合的驴抗豚鼠抗体(Jackson ImmunoResearch Laboratories,目录号:106-075-003)
  14. Alexa 350结合的山羊抗兔抗体(Life Technologies,Invitrogen TM ,目录号:A-21068)
  15. FITC缀合的山羊抗小鼠抗体(Jackson ImmunoResearch Laboratories,目录号:715-095-166)
  16. ProLong Gold Antifade Reagent(Life Technologies,Invitrogen TM ,目录号:P36935)
  17. 10x PBS(Gibco ,目录号:70011-044)
  18. 阻止解决方案(参见配方)

设备

  1. 玻璃瓶
  2. 加湿室
  3. 共焦显微镜
  4. 组织盒

程序

  1. 福尔马林固定
    1. 在4℃下旋转的同时,通过10ml 10%福尔马林溶液在15ml锥形管中固定过夜胰腺(〜5mm×〜5mm)。
    2. 第二天,将固定的组织转移到组织盒,并将其存储在70%乙醇溶液中,直到在室温下制备石蜡块(至少1天)。
    3. 制作固定组织的石蜡块。
    4. 使用Microtome制作石蜡块的滑块。

  2. 滑膜切片的免疫染色
    1. 脱蜡步骤
      1. 在99.5%二甲苯溶液中保持切片5分钟2次
      2. 保持切片在99.5%二甲苯溶液中10分钟2次
      3. 用新玻璃瓶将切片在100%乙醇中保持3分钟
      4. 保持切片在95%乙醇中3分钟
      5. 保持切片在85%乙醇中3分钟。
      6. 保持切片在70%乙醇中3分钟。
      7. 保持切片在50%乙醇中5分钟。
      8. 用新的玻璃罐将切片在0.85%NaCl中保持5分钟
      9. 在1x PBS中保持切片5分钟。
    2. 渗透步骤
      1. 在室温(RT)下,在含有新玻璃广口瓶的0.1%Triton-X 100的PBS中孵育切片30分钟。
      2. 孵育切片5分钟,在1x PBS与新玻璃瓶3次。
    3. 阻断,一级和二级抗体(Ab)结合步骤
      1. 在组织周围吸气,直到幻灯片,而不是组织,是干燥的。 仔细地,用油脂窒息在组织周围。
      2. 在coplin广口瓶中用含有0.05%Tween 20的PBS中的2%BSA封闭30分钟
      3. 吸出阻断缓冲液并在含有0.05%Tween 20的PBS中的2%BSA中在4℃下在潮湿的胃室中用胰高血糖素原始Ab(1/100稀释)孵育切片过夜(o/n)。使用足够的抗体溶液(〜150微升)完全淹没的部分。
      4. 吸出一抗,并在含有0.05%Tween 20的PBS中的2%BSA中洗涤切片5分钟,在coplin广口瓶中洗涤2次。
      5. 吸出洗涤缓冲液,并在加湿室中与相应的二抗Alexa350共轭的山羊抗兔抗体(1/500稀释)孵育切片1.15小时,在室温黑暗中。在含有0.05%Tween 20的PBS中的2%BSA中稀释辅助Ab。使用足够的抗体溶液(〜150μl)完全浸没切片。
      6. 吸出第二抗体,并在含有0.05%吐温20的PBS中的2%BSA中洗涤切片5分钟,在Coplin瓶中2次。在组织周围吸气,直到幻灯片,而不是组织,是干燥的。并在具有胰岛素初级Ab(1/500稀释)的湿润室中在含有0.05%Tween 20的PBS中的2%BSA中在黑暗中在室温孵育切片1.5小时。使用足够的抗体溶液(〜150微升)完全淹没的部分。
      7. 吸出第一抗体,并在含有0.05%Tween 20的PBS中的2%BSA中在室温下在coplin瓶中黑暗中洗涤切片5分钟。
      8. 在组织周围吸气,直到幻灯片,而不是组织,是干燥的。并在加湿的室中与相应的次级德克萨斯红缀合的驴抗豚鼠Ab孵育切片在室温在黑暗中1小时。在含有0.05%Tween 20的PBS中的2%BSA中稀释辅助Ab(1/1000稀释)。使用足够的抗体溶液(〜150μl)完全浸没切片。
      9. 吸出第二抗体,并在含有0.05%Tween 20的PBS中的2%BSA中在室温下在暗处在coplin瓶中洗涤切片5分钟。在组织周围吸气,直到幻灯片,而不是组织,是干燥的。并在加湿的外倾中用EGFP一抗(1/500稀释)孵育切片,在暗处4℃下孵育1小时。在含有0.05%Tween 20的PBS中的2%BSA中稀释的原代Ab。使用足够的抗体溶液(〜150μl)完全浸没切片。
      10. 吸出第一抗体,并在含有0.05%Tween 20的PBS中的2%BSA中在室温下在coplin瓶中黑暗中洗涤切片5分钟。
      11. 吸出洗涤缓冲液,孵育在湿润室与相应的二级FITC结合的山羊抗小鼠Ab(1/500稀释)在室温下在黑暗中1小时的切片。在含有0.05%Tween 20的PBS中的2%BSA中稀释辅助Ab。使用足够的抗体溶液(〜150μl)完全浸没切片。
      12. 吸出第二抗体,并在含有0.05%吐温20的PBS中的2%BSA中在室温下在coplin瓶中黑暗中洗涤切片5分钟。
  3. 安装步骤
    1. 在组织周围抽吸,直到幻灯片,而不是组织,是干的,添加〜50微升含有DAPI的ProLong Gold Antifade Reagent到每个部分,并在截面上放置盖玻片。
      注意:小心,以避免损坏组织,滑动太多的盖玻片,不要引入气泡。 吸出过量的试剂。
    2. 干燥部分在室温下数小时或过夜避光。
    3. 一旦幻灯片完全干燥,用清洁的指甲油密封盖玻片的边缘。 载玻片可储存于-20°C,避光照射数周。

食谱

  1. 封闭溶液(含有0.05%Tween20的PBS pH7.4的2%BSA) 10 g BSA
    50ml 10×PBS
    0.25ml Tween20 将ddH 2 O添加到500 ml

代表数据



图1.胰腺组织切片中EGFR,胰岛素和胰高血糖素的三重免疫染色在谭施用后3周从小鼠获得胰组织切片,以从转基因诱导EGFP。胰腺组织切片对EGFP(绿色),胰岛素(红色)和胰高血糖素(蓝色)进行三重免疫染色,并且单独显示代表性单通道荧光图像并合并(每组的右下图像)。 [请引用参考文献1(图1B)]

致谢

这项工作部分得到教育,科学和技术赠款部2011-0011433,2012M3A9C3048686和2014R1A1A4A01004329(S.H.B)资助的韩国国家研究基金会和NIH授予DK42394,HL52173和HL057346(R.J.K.)的部分支持。 R.J.K.是霍华德休斯医学研究所的研究员。该协议改编自Back等人(2009),并且适应的协议的第一简短版本在Han等人(2013)中公开。

参考文献

  1. Back,S.H. *,Scheuner,D. *,Han,J.,Song,B.,Ribick,M.,Wang,J.,Gildersleeve,R.D.,Pennathur,S.and Kaufman,R.J。(2009)。 翻译 通过eIF2alpha磷酸化的衰减防止氧化应激和维持β细胞中的分化状态。细胞Metab 10(1):13-26。 *这些作者同样对这项工作贡献了
  2. 汉族,哈佛大学学报(社会科学版),北京中医药大学学报(医学版),北京中医药大学学报 ,MS,Sartor,MA和Kaufman,RJ(2013)。 ER应激诱导的转录调节增加导致细胞死亡的蛋白质合成。 Nat Cell Biol 15(5):481-490。 *这些作者同样对这项工作贡献了
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Choi, W., Kaufman, R. J. and Back, S. H. (2014). TRIPLE (Insulin, Glucagon and EGFP) Immunofluorescence Staining Protocol in Pancreas. Bio-protocol 4(5): e1056. DOI: 10.21769/BioProtoc.1056.
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