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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.

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

免疫学 > 免疫细胞染色 > 免疫检测
作者: Zheng Liu
Zheng LiuAffiliation: The Feinstein Institute for Medical Research, Manhasset, NY, USA
For correspondence: zl2119@caa.columbia.edu
Bio-protocol author page: a12
Anne Davidson Lab, 5/5/2011, 9214 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.65

[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)

[Abstract] 无论是在狼疮患者体内还是易患狼疮的小鼠体内,肾脏免疫复合物的沉积和白细胞渗透都是狼疮肾脏炎的特征。这个实验步骤描述了如何对小鼠肾脏的冷冻切片进行染色然后方用荧光显微镜来研究这些特征。它是由范斯坦医学研究院安妮˙戴维森博士实验室修改完善的。

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.

材料和试剂

 

I. 抗体

1.        兔抗鼠IgG2a FITC (Southern Biotech 1155-02)

2.        兔抗鼠 IgG3 FITC (Southern Biotech 1190-02)

3.        兔抗鼠IgD PE (Southern Biotech 1120-09)

4.        兔抗鼠F480 PE (Invitrogen MF48004)

5.        兔抗鼠B220 PE (BD Pharmingen BD553090)

6.        仓鼠抗鼠CD11c PE (BD Pharmingen BD553802)

7.        兔抗鼠 CD4 PE (BD Pharmingen BD553049)

注意: 上述抗体都被作者验证过,也有可能根据作者的需要被其他抗体替换过.

 

II.其它材料:

8.        丙酮 (Sigama 650501-4L)

9.        Tek O.C.T. Compound (Sakura Finetek 4587)

10.    3% FBS (Fetal Bovine Serum, Sigma, F2442-500ML) in PBS.

11.    封闭液: 0.5% Mouse BD Fc Block (BD Pharmingen 553141) in 3% FBS

12.    DAPI nucleic acid stain (Invitrogen D1306)

13.    Glycergel Mounting Medium (Dako C0563)

14.    玻璃染色缸(Cole-Parmer EW-48585-00)

15.    ImmEdge Pen (Vector Laboratories, Inc, H-4000)

 

步骤

 

1.        鼠的组织需要在Tek O.C.T.里速冻.Compound并切成5μM切片.

2.        -80oC 取出载玻片,常温下在黑暗放置直到融化.

3.        通风橱中将载玻片在丙酮中浸泡5分钟并且丙酮需要在-20oC 下预冷.

4.        PBS 中浸泡载玻片5分钟.

5.        重复步骤4

6.        在常温下干燥载玻片并且用an ImmEdge pen 圈组织

7.        每个组织加入大约100uL封闭液并且确保溶液在这个圈里并且整个组织都被覆盖.

8.        在室温下孵育30分钟.

9.        干燥载玻片.注意: 不要过度干燥载玻片.

10.    3% FBS/PBS 中稀释所要的抗体1/50.

11.    向每个组织的载玻片中加入100uL稀释的抗体.

12.    在室温下黑暗孵育载玻片1小时

13.    PBS洗载玻片3次(每次5分钟). 保持黑暗.

14.    向每个组织的载玻片中加入100uL DAPI solution (300nM).

15.    在常温下培养载玻片1-5分钟. 保持黑暗.

16.    PBS洗载玻片3次(每次5分钟). 保持黑暗.

17.    在水浴锅里加热Glycergel 包埋剂到65度,直到融化.

18.    从载玻片里排出PBS.

19.    在载玻片的顶部加入包埋剂,用盖玻片盖上组织,确保没有生成气泡.

20.    载玻片弄好之后马上去看荧光显微镜或者在黑暗4oC 下保存以后用.

注意:这个方法被成功的用于染色鼠的肾脏免疫复合体,肾白细胞infiltrates(CD4 T cells, B220 B cells, F480 macrophages, and CD11c dendritic cells),鼠的脾组织比如B cell follicles (IgD+) germinal centers (Peanut Agglutinin+)

 

参考 文献

 

1.        Liu Z., Bethunaickan R., Huang W., Lodhi U., Solano I., Madaio M.P., Davidson A. (2011). Interferon-alpha accelerates murine systemic lupus erythematosus in a T cell-dependent manner. Arthritis and Rheumatism 63(1): 219-29. 

2.        Ramanujam M., Wang X., Huang W., Liu Z., Schiffer L., Tao H., Frank D., Rice J., Diamond B., Yu K.O., Porcelli S., Davidson A. (2006). Similarities and differences between selective and nonselective BAFF blockade in murine SLE. Journal of Clinical Investigation 116(3): 724-34. 

 

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How to cite this protocol: Liu, Z. (2011). Immunofluorescent Staining of Murine Tissue. Bio-protocol Bio101: e65. DOI: 10.21769/BioProtoc.65; Full Text



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