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[Bio101] Immunofluorescent Staining of Murine Tissue
[Bio101] 鼠源组织的免疫荧光染色

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Abstract

Renal immune complex deposition and leukocyte infiltration are characteristic of lupus nephritis in human patients and lupus-prone mice. This protocol describes how to stain frozen sections of murine kidney to study these features using fluorescent microscopy. This protocol was developed or modified in Dr. Anne Davidson’s lab at Feinstein Institute for Medical Research.

Keywords: Lupus(系统性红斑狼疮), Nephrtitis(nephrtitis), Mouse(鼠标), Immunofluorescent(免疫荧光), Frozen(冷冻)

Materials and Reagents

  1. Antibodies
    1. Rat anti-mouse IgG2a FITC (Southern Biotech, catalog number: 1155-02 )
    2. Rat anti-mouse IgG3 FITC (Southern Biotech, catalog number: 1190-02 )
    3. Rat anti-mouse IgD PE (Southern Biotech, catalog number: 1120-09 )
    4. Rat anti-mouse F480 PE (Life Technologies, Invitrogen™, catalog number: MF48004 )
    5. Rat anti-mouse B220 PE (BD Biosciences, Pharmingen™, catalog number: BD553090 )
    6. Hamster anti-mouse CD11c PE (BD Biosciences, Pharmingen™, catalog number: BD553802 )
    7. Rat anti-mouse CD4 PE (BD Biosciences, Pharmingen™, catalog number: BD553049 )
      Note: The above antibodies have been tested by the author and may be substituted with the antibodies desired by users.

  2. Other materials
    1. Murine kidney tissues
    2. Acetone (Sigma- Aldrich, catalog number: 650501-4L )
    3. Tek O.C.T. Compound (Sakura Finetek, catalog number: 4587 )
    4. 3% Fetal bovine serum (FBS) (Sigma- Aldrich, catalog number: F2442-500ML ) in PBS.
    5. 0.5% Mouse BD Fc Block (BD Biosciences, Pharmingen™, catalog number: 553141 )
    6. DAPI nucleic acid stain (Life Technologies, Invitrogen™, catalog number: D1306 )
    7. Glycergel mounting medium (Dako, catalog number: C0563 )
    8. Block solution

Equipment

  1. Glass staining jars (Cole-Parmer, catalog number: EW-48585-00 )
  2. ImmEdge Pen (Vector Laboratories, Inc, catalog number: #H-4000 )

Procedure

  1. Murine tissues need to be snap frozen in Tek O.C.T. compound and cut into 5 μM sections.
  2. Remove slides from -80 °C and keep in the dark at room temperature (RT) until thawed.
  3. In the fume hood, immerse slides in acetone for 5 min and acetone needs to be pre-chilled in -20 °C.
  4. Immerse slides in PBS for 5 min.
  5. Repeat step 4.
  6. Dry slides at RT and circle tissues with an ImmEdge pen.
  7. Apply ~100 μl blocking solution per tissue and make sure that the solution is within the circles and the entire tissue is covered.
  8. Incubate at RT for 30 min.
  9. Dry the slides.
    Note: Do not dry the slides excessively.
  10. Dilute the desired antibody 1/50 in 3% FBS/PBS.
  11. Apply 100 μl antibody dilution per tissue to the slides.
  12. Incubate the slides for 1 h at RT. Keep in dark.
  13. Wash slides in PBS three times (5 min each wash). Keep in dark.
  14. Apply 100 μl DAPI solution (300 nM) per tissue to the slides.
  15. Incubate the slides at RT for 1-5 min. Keep in dark.
  16. Wash the slides in PBS three times (5 min each). Keep in dark.
  17. Heat glycergel mounting medium to 65 °C in a water-bath until melted.
  18. Drain out the PBS from the slides.
  19. Apply the mounting solution to the top of the slides and cover tissue with cover glass, making sure no air bubbles are formed.
  20. Slides are now ready for immediate use for fluorescent microscopy or can be stored at 4 °C in dark for future use.

Notes

This protocol has been successfully used for staining murine renal immune complexes, renal leukocyte infiltrates (CD4 T cells, B220 B cells, F480 macrophages, and CD11c dendritic cells), and murine splenic structures such as B cell follicles (IgD+) germinal centers (Peanut Agglutinin+).

Recipes

  1. Block solution
    0.5% Mouse BD Fc Block in 3% FBS

Acknowledgments

This protocol was developed or modified in Dr. Anne Davidson’s lab at Feinstein Institute for Medical Research, NY, USA. This work was supported by grants from the NY SLE Foundation (RB), Rheuminations, NIH AI082037 and AR 049938-01, NIH (PO1 AI51392 and the Flow Cytometry and Protein Expression and Tetramer Cores of PO1 AI51392).

References

  1. Liu, Z., Bethunaickan, R., Huang, W., Lodhi, U., Solano, I., Madaio, M. P. and Davidson, A. (2011). Interferon-alpha accelerates murine systemic lupus erythematosus in a T cell-dependent manner. Arthritis Rheum 63(1): 219-229.
  2. Ramanujam, M., Wang, X., Huang, W., Liu, Z., Schiffer, L., Tao, H., Frank, D., Rice, J., Diamond, B., Yu, K. O., Porcelli, S. and Davidson, A. (2006). Similarities and differences between selective and nonselective BAFF blockade in murine SLE. J Clin Invest 116(3): 724-734.

简介

肾脏免疫复合物沉积和白细胞浸润是人类患者和易患狼疮的小鼠的狼疮肾炎的特征。 这个协议描述如何染色鼠肾脏的冷冻切片,以研究这些功能使用荧光显微镜。 这个协议是在费恩斯坦医学研究所的Anne Davidson博士的实验室中开发或修改的。

关键字:系统性红斑狼疮, nephrtitis, 鼠标, 免疫荧光, 冷冻

材料和试剂

  1. 抗体
    1. 大鼠抗小鼠IgG2a FITC(Southern Biotech,目录号:1155-02)
    2. 大鼠抗小鼠IgG3 FITC(Southern Biotech,目录号:1190-02)
    3. 大鼠抗小鼠IgD PE(Southern Biotech,目录号:1120-09)
    4. 大鼠抗小鼠F480PE(Life Technologies,Invitrogen TM,目录号:MF48004)
    5. 大鼠抗小鼠B220PE(BD Biosciences,Pharmingen TM,目录号:BD553090)
    6. 仓鼠抗小鼠CD11c PE(BD Biosciences,Pharmingen TM,目录号:BD553802)
    7. 大鼠抗小鼠CD4PE(BD Biosciences,Pharmingen TM,目录号:BD553049)
      注意:上述抗体已经被作者测试,可能被用户需要的抗体替代。

  2. 其他材料
    1. 鼠肾组织
    2. 丙酮(Sigma-Aldrich,目录号:650501-4L)
    3. Tek O.C.T. 化合物(Sakura Finetek,目录号:4587)
    4. 3%PBS中的胎牛血清(FBS)(Sigma-Aldrich,目录号:F2442-500ML)。
    5. 0.5%小鼠BD Fc Block(BD Biosciences,Pharmingen TM,目录号:553141)
    6. DAPI核酸染色(Life Technologies,Invitrogen TM,目录号:D1306)
    7. Glycergel封固剂(Dako,目录号:C0563)
    8. 封锁解决方案

设备

  1. 玻璃染色罐(Cole-Parmer,目录号:EW-48585-00)
  2. ImmEdge Pen(Vector Laboratories,Inc,目录号:#H-4000)

程序

  1. 鼠科组织需要在Tek O.C.T中快速冻结。 复合并切成5μM切片
  2. 从-80°C取出载玻片,在室温(RT)下保持黑暗,直到解冻
  3. 在通风橱中,将载玻片浸没在丙酮中5分钟,并且丙酮需要在-20℃预冷。
  4. 在PBS中浸泡幻灯片5分钟。
  5. 重复步骤4.
  6. 使用ImmEdge笔在RT和环组织处干燥载玻片。
  7. 每个组织应用〜100μl阻断溶液,并确保溶液在圆内,整个组织被覆盖
  8. 在室温下孵育30分钟。
  9. 干燥幻灯片。
    注意:请勿过度干燥幻灯片。
  10. 在3%FBS/PBS中稀释所需抗体1/50
  11. 每个组织应用100微升抗体稀释到幻灯片
  12. 在室温下孵育载玻片1小时。 保持在黑暗中。
  13. 洗涤PBS中的幻灯片三次(每次洗涤5分钟)。 保持在黑暗中。
  14. 应用100微升DAPI溶液(300 nM)每个组织到幻灯片
  15. 孵育幻灯片在室温下1-5分钟。 保持在黑暗中。
  16. 在PBS中洗涤载玻片三次(每次5分钟)。 保持在黑暗中。
  17. 将聚乙烯基封固剂在水浴中加热至65℃,直至熔融
  18. 从幻灯片中排出PBS。
  19. 将安装溶液涂抹在载玻片的顶部,并用盖玻片覆盖组织,确保没有气泡形成
  20. 幻灯片现在准备立即用于荧光显微镜或可以存储在4°C在黑暗中以备将来使用。

笔记

该方案已经成功地用于染色鼠肾免疫复合物,肾白细胞浸润(CD4T细胞,B220B细胞,F480巨噬细胞和CD11c树突细胞)和鼠脾结构如B细胞滤泡(IgD < /生发中心(Peanut Agglutinin )。

食谱

  1. 封锁解决方案
    0.5%小鼠BD Fc Block在3%FBS中

致谢

该方案在Anne Davidson博士在Feinstein Institute for Medical Research,NY,USA的实验室中开发或修改。 这项工作得到NY SLE基金会(RB),Rheuminations,NIH AI082037和AR 049938-01,NIH(PO1 AI51392和PO1 AI51392的流式细胞术和蛋白质表达和四聚体核)的赠予的支持。

参考文献

  1. Liu,Z.,Bethunaickan,R.,Huang,W.,Lodhi,U.,Solano,I.,Madaio,M.P.and Davidson,A。(2011)。 干扰素-α以T细胞依赖的方式加速小鼠系统性红斑狼疮。 arthritis Rheum 63(1):219-229。
  2. Ramanujam,M.,Wang,X.,Huang,W.,Liu,Z.,Schiffer,L.,Tao,H.,Frank,D.,Rice,J.,Diamond,B.,Yu,KO,Porcelli ,S.and Davidson,A。(2006)。 鼠科SLE中选择性和非选择性BAFF阻断之间的相似性和差异。 Clin Invest 116(3):724-734
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引用:Liu, Z. (2011). Immunofluorescent Staining of Murine Tissue. Bio-protocol Bio101: e65. DOI: 10.21769/BioProtoc.65;
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