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Colon Tissue Immunoelectron Microscopy
结肠组织免疫电镜分析   

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Abstract

The method described here is intended to study intracellular localization of proteins in colon cells. This protocol was used to localize IRE1β in the endoplasmic reticulum membrane. We used anti-IRE1β antibody raised in guinea pig for this purpose. We also studied the location of BiP (also known as GRP78), with the antibody raised in rabbit. Both antibodies used with appropriate gold particle-conjugated secondary antibodies gave good results. Primary mouse antibodies are not recommended because secondary anti-mouse antibodies also react with the internal mouse epitopes.

Keywords: Immunoelectron microscopy(免疫电镜), Goblet cells(杯状细胞), ER stress(内质网应激), IRE1(IRE1), BiP(BIP)

Materials and Reagents

  1. Mice
  2. Paraformaldehyde (TAAB, catalog number: P001/1 )
  3. Sucrose
  4. Glutaraldehyde (Polysciences, catalog number: 111-30-8 )
  5. Ethanol
  6. Propylene oxide
  7. LR White (London resin, medium)
  8. Hydrogen peroxide
  9. ImmunoSaver (Nisshin EM, catalog number: 333 )
  10. Gelatin
  11. Tween 20
  12. Uranyl acetate
  13. Distilled water or Milli-Q water
  14. Primary antibodies
  15. 10-nm gold-conjugated secondary antibodies (we purchased from EY laboratories in Tsuru et al., 2013)
  16. Phosphate buffered saline (PBS) (see Recipes)
  17. 0.1 M phosphate buffer (pH 7.2) (see Recipes)
  18. Antigen-retrieval solution (see Recipes)

Equipment

  1. Peristaltic pump
  2. Refrigerator
  3. Freezer
  4. UV irradiator (Nisshin EM, model: TUV-100 ) with 15 W UV light
  5. Gold grid (Maxtaform HR25)
  6. Ultra microtome (Leica Microsystems, model: Ultracut UCT )
  7. Diamond knife (Diatome AG, catalog number: ultra 45 )
  8. Incubator
  9. Microwave (Nisshin EM, catalog number: MWF-2 )
  10. Electron microscope (Hitachi, model: H-7100 )

Procedure

  1. Sample preparation
    1. Fix whole mice by perfusion.
      1. Set up perfusion system consisted of needle, tubing, peristaltic pump and beakers with PBS (ice-cold) or 4% paraformaldehyde in PBS (ice-cold).
      2. Anesthetize mice and expose hearts by surgery.
      3. Insert the needle into the left ventricle and make a hole in the right atrium.
      4. Remove blood from mice by perfusion (8 ml/min) with PBS (ice-cold).
      5. Fix mice by perfusion (8 ml/min) with 4% paraformaldehyde in PBS (ice-cold).
    2. Isolate colons and cut them up into small pieces (~1.5 mm thick).
    3. Fix again with 0.1% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) for 2 h at 4 °C.
    4. Wash samples by 0.1 M phosphate buffer (pH 7.2) containing 8% sucrose.
    5. Dehydrate samples with a series of ethanol as follows.
      1. 25% ethanol, 10 min at 4 °C
      2. 60% ethanol, 30 minu at -30 °C
      3. 80% ethanol, 30 min at -30 °C
      4. 99% ethanol, 30 min at -30 °C
      5. 99% ethanol, 10 min at RT
      6. 100% ethanol, two changes, 10 min each at RT
    6. To promote the infiltration of resin, remove ethanol with propylene oxide as follows.
      1. Ethanol-propylene oxide (2:1), 10 min at RT
      2. Ethanol-propylene oxide (1:1), 10 min at RT
      3. Ethanol-propylene oxide (1:2), 10 min at RT
      4. Propylene oxide, 20 minutes at RT
    7. Make resin infiltrate into samples by immersion as follows.
      1. Propylene oxide-LR White containing accelerator (3:1), 2 h at RT
      2. Propylene oxide-LR White containing accelerator (1:1), 2 h at RT
      3. Propylene oxide-LR White containing accelerator (1:3), 2 h at RT
      4. LR White containing accelerator, 12 h at RT
    8. Polymerize resin under UV irradiation for two days at RT.
    9. Cut the embedded samples (90 nm thick) with ultra microtome and mount on uncoated gold grids (300 mesh).

  2. Immunostaining
    1. Treat sections with 3% (vol/vol) hydrogen peroxide for 30 min at RT to unmask epitope.
    2. Wash sections with distilled water.
    3. Treat sections with antigen-retrieval solution for 15 min under microwave at 95 °C by interval of 2 sec.
    4. Block sections with 0.1% gelatin in PBST (PBS containing 0.05% Tween 20) for 30 min at 37 °C.
    5. Reacted sections with primary antibodies diluted with PBST overnight at RT.
    6. Wash sections three times with PBST for 10 min each at RT.
    7. React sections with 10-nm gold-conjugated secondary antibodies diluted with PBST for 2 h at RT.
    8. Wash sections twice with PBST for 10 min each at RT.
    9. Wash sections twice with distilled water for 5 min each at RT.
    10. Stain sections with 4% uranyl acetate for 10 min at RT.
    11. Wash sections with distilled water.
    12. Observe sections with an electron microscope.

Recipes

  1. Phosphate buffered saline (PBS)
    Dissolve 2.89 g of Na2HPO4·12H2O, 0.2 g of KH2PO4, 8.0 g of NaCl, and 0.2 g of KCl in distilled water
    Adjust the final volume to 1,000 ml
    Sterilize by autoclaving
  2. 0.1 M phosphate buffer (pH 7.2)
    Solution A: Dissolve 13.61 g KH2PO4 in 1,000 ml distilled water
    Solution B: Dissolve 17.8 g Na2HPO4·2H2O in 1,000 ml distilled water
    Mix appropriate volumes of solution A and solution B to adjust pH to 7.2
  3. Antigen-retrieval solution
    Dilute ImmunoSaver 200-fold with distilled water

Acknowledgments

This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grants 24228002, 24248019 (to K.K.), and 14580699 (to A.T.), Ministry of Education, Culture, Sports, Science and Technology in Japan (MEXT) KAKENHI Grant 19058010 (to K.K.), The Uehara Memorial Foundation (K.K.), Takeda Science Foundation (K.K.), Mitsubishi Foundation (K.K.).

References

  1. Tsuru, A., Fujimoto, N., Takahashi, S., Saito, M., Nakamura, D., Iwano, M., Iwawaki, T., Kadokura, H., Ron, D. and Kohno, K. (2013). Negative feedback by IRE1β optimizes mucin production in goblet cells. Proc Natl Acad Sci U S A 110(8): 2864-2869.

简介

本文所述的方法旨在研究结肠细胞中蛋白质的细胞内定位。 该方案用于定位IRE1β在内质网膜中。 我们使用在豚鼠中产生的抗IRE1β抗体用于此目的。 我们还研究了在兔中产生的抗体的BiP(也称为GRP78)的位置。 与合适的金颗粒缀合的二抗一起使用的两种抗体都给出了良好的结果。 不推荐初级小鼠抗体,因为次级抗小鼠抗体也与内部小鼠表位反应。

关键字:免疫电镜, 杯状细胞, 内质网应激, IRE1, BIP

材料和试剂

  1. 小鼠
  2. 多聚甲醛(TAAB,目录号:P001/1)
  3. 蔗糖
  4. 戊二醛(Polysciences,目录号:111-30-8)
  5. 乙醇
  6. 环氧丙烷
  7. LR White(伦敦树脂,中型)
  8. 过氧化氢
  9. ImmunoSaver(Nisshin EM,目录号:333)
  10. 明胶
  11. 吐温20
  12. 乙酸乙烯酯
  13. 蒸馏水或Milli-Q水
  14. 一抗
  15. 10-nm金共轭二抗(我们从Tsuru等人的EY实验室购买,2013)
  16. 磷酸盐缓冲盐水(PBS)(见Recipes)
  17. 0.1 M磷酸盐缓冲液(pH 7.2)(参见配方)
  18. 抗原检索溶液(参见配方)

设备

  1. 蠕动泵
  2. 冰箱
  3. 冰柜
  4. UV辐照器(Nisshin EM,型号:TUV-100),用15W紫外光
  5. 金格(Maxtaform HR25)
  6. 超薄切片机(Leica Microsystems,型号:Ultracut UCT)
  7. 金刚石刀(Diatome AG,目录号:ultra 45)
  8. 孵化器
  9. 微波(Nisshin EM,目录号:MWF-2)
  10. 电子显微镜(日立,型号:H-7100)

程序

  1. 样品制备
    1. 通过灌注固定整个小鼠。
      1. 用PBS(冰冷)或PBS中的4%多聚甲醛(冰冷的)设置由针,管,蠕动泵和烧杯组成的灌注系统。
      2. 麻醉小鼠和手术暴露心脏。
      3. 将针插入左心室,并在右心房中打孔。
      4. 通过用PBS(冰冷)灌注(8ml/min)从小鼠中除去血液
      5. 通过用含4%多聚甲醛的PBS(冰冷的)灌注(8ml/min)固定小鼠
    2. 隔离冒号,切成小块(约1.5毫米厚)。
    3. 再次用0.1%戊二醛和4%多聚甲醛在0.1M磷酸盐缓冲液(pH 7.2)中在4℃固定2小时。
    4. 用含有8%蔗糖的0.1M磷酸盐缓冲液(pH7.2)洗涤样品
    5. 用如下的一系列乙醇脱水样品
      1. 25%乙醇,在4℃下10分钟
      2. 60%乙醇,-30℃下30分钟,
      3. 80%乙醇,-30℃下30分钟
      4. 99%乙醇,-30℃下30分钟
      5. 99%乙醇,RT下10分钟
      6. 100%乙醇,两次更换,每次10分钟,在室温下
    6. 为了促进树脂的渗透,如下用环氧丙烷除去乙醇。
      1. 乙醇 - 环氧丙烷(2:1),室温下10分钟
      2. 乙醇 - 环氧丙烷(1:1),室温下10分钟
      3. 乙醇 - 环氧丙烷(1:2),RT下10分钟
      4. 环氧丙烷,室温下20分钟
    7. 使树脂通过浸渍如下浸入样品中。
      1. 环氧丙烷LR含白色促进剂(3:1),室温下2小时
      2. 环氧丙烷LR含白色促进剂(1:1),室温下2小时
      3. 环氧丙烷LR含白色促进剂(1:3),室温下2小时
      4. LR白色含促进剂,室温下12小时
    8. 在室温下在UV照射下聚合树脂两天
    9. 用超薄切片机切割嵌入的样品(90nm厚),并安装在未涂层的金网格(300目)上。

  2. 免疫染色
    1. 用3%(体积/体积)过氧化氢在室温处理切片30分钟,以暴露表位
    2. 用蒸馏水清洗切片。
    3. 用抗原回收溶液在微波下在95℃下以2秒的间隔处理切片15分钟
    4. 用0.1%明胶在PBST(含有0.05%Tween 20的PBS)中在37℃封闭切片30分钟
    5. 反应切片,用PBST在室温下稀释一次抗体过夜
    6. 用PBST洗涤切片三次,每次10分钟
    7. 反应切片用10-nm金共轭的二抗在PBST稀释2小时在室温
    8. 用PBST洗涤切片两次,每次10分钟
    9. 用蒸馏水洗涤切片两次,每次5分钟
    10. 在室温下用4%乙酸双氧铀染色10分钟
    11. 用蒸馏水清洗切片。
    12. 用电子显微镜观察切片。

食谱

  1. 磷酸盐缓冲盐水(PBS)
    将2.89g的Na 2 HPO 4·12H 2 O,0.2g的KH 2 PO 4·6H 2 O, 4,8.0g NaCl和0.2g KCl的蒸馏水中 将最终体积调整为1,000 ml
    高压灭菌
    灭菌
  2. 0.1M磷酸盐缓冲液(pH 7.2) 溶液A:将13.61g KH 2 PO 4溶解在1,000ml蒸馏水中
    溶液B:在1,000ml蒸馏水中溶解17.8g Na 2 HPO 4·2H 2 2H 2 O
    混合适当体积的溶液A和溶液B,以将pH调节至7.2
  3. 抗原检索溶液
    用蒸馏水稀释ImmunoSaver 200倍

致谢

这项工作由日本科学促进会(JSPS)KAKENHI批准24228002,24248019(KK)和14580699(AT),日本教育,文化,体育,科学和技术部(MEXT)KAKENHI 格兰特19058010(KK),上原纪念基金会(KK),武田科学基金会(KK),三菱基金会(KK)。

参考文献

  1. Tsuru,A.,Fujimoto,N.,Takahashi,S.,Saito,M.,Nakamura,D.,Iwano,M.,Iwawaki,T.,Kadokura,H.,Ron,D.and Kohno, 2013)。 IRE1β的负反馈优化杯状细胞中的粘蛋白产生。 Proc Natl Acad Sci USA 110(8):2864-2869。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Iwano, M., Tsuru, A. and Kohno, K. (2013). Colon Tissue Immunoelectron Microscopy. Bio-protocol 3(20): e932. DOI: 10.21769/BioProtoc.932.
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