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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

WC Woogyun Choi
Randal J. Kaufman Randal J. Kaufman
Sung Hoon Back Sung Hoon Back

发布: 2014年03月05日第4卷第5期 DOI: 10.21769/BioProtoc.1056 浏览次数: 15784

评审: Lin FangFanglian He

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Cover of Nature Cell Biology, featuring study using the protocol.

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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

English
中文翻译

文章信息

版权信息

© 2014 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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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分类

细胞生物学 > 细胞成像 > 荧光

生物化学 > 蛋白质 > 免疫检测 > 免疫染色法

细胞生物学 > 细胞染色 > 蛋白质

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