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ImmunoFISH for Mice and Baboons Frozen Sections
小鼠和狒狒冰冻切片的ImmunoFISH试验   

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参见作者原研究论文

本实验方案简略版
Nature Cell Biology
Apr 2012

Abstract

This protocol is optimized for immunoFISH staining of OCT section of mouse tissues. It combines immunofluorescence for DNA damage response factors (e.g. 53BP1) (Le et al., 2010) and FISH against telomeric DNA.

Keywords: ImmunoFISH (immunoFISH), DNA damage response (DNA损伤反应), Telomeres (端粒酶), Baboon tissues (Baboon组织)

Materials and Reagents

  1. Tissue
  2. OCT
  3. 4% formaldehyde
  4. PBS
  5. Triton
  6. Goat serum
  7. BSA
  8. Primary antibody: 53BP1 #NB 100-304 rabbit from Novus
  9. Second antibody: goat anti-rabbit Alexa Fluor® 488 Dye
  10. Triton
  11. Glycine
  12. Mowiol 4-88 reagent (Calbiochem®)
  13. Formamide
  14. Tris HCl, pH 7.4
  15. Telomeric PNA probe (TelC-Cy3 from PANAGENE, catalog number: F1002-5 )
  16. Tween-20
  17. DAPI
  18. Hybridization mixture (see Recipes)
  19. Blocking reagent (Roche Diagnostics, catalog number: 11096176001 ) (see Recipes)
  20. Wash solution I (see Recipes)
  21. Wash solution II (see Recipes)

Equipment

  1. Glass slide
  2. Metal thermoblock
  3. Humidified chamber

Procedure

  1. Frozen tissue placed in OCT without fixation.
  2. When needed, slice to the desired thickness (8-10 micron), dry the slides few minutes (often the time to prepare the other slides) and freeze again at -80 °C.
  3. The day of the staining, thaw the slides and fix for 20 min in 4% formaldehyde.
  4. Wash slides with PBS for 3 x 5 min at RT.
  5. Permeabilize slides with 0.5% Triton in PBS for 5 min at RT.
  6. Wash 2x with PBS 5 min at RT.
  7. Block in 5% Goat serum diluted in PBS + 1% BSA for 60 min.
  8. Incubate at 4 °C: 53BP1 #NB 100-304 (rabbit from Novus) 1:100 in PBS, 2.5% goat serum, 1% BSA. Use 60-80 μl for each slide.
  9. Wash once quickly and 3 x 10 min with PBS at RT.
  10. Secondary: goat anti-rabbit (Alexa 488) (1/100) in PBS + 1% BSA for 60 min at RT.
  11. Wash once quickly and 3 x 10 min with PBS RT.
  12. Re-fix tissue with PFA 4% + Triton 0.1%, 10 min RT.
  13. Incubate with glycine 10 mM in H2O, 30 min, RT.
  14. Wash with 1x PBS, 3 times, 5 min.
  15. Prepare the hybridization mixture and put 30-50 μl directly on the sample.
  16. Put a glass slide carefully on the drop without making bubbles.
  17. Put the slide directly on a metal thermoblock at 80 °C, 5 min.
  18. Hybridize in a humidified chamber, 2 h, RT.
  19. Remove glass from the slide.
  20. Wash with Wash solution I, twice, 15 min.
  21. Wash with Wash solution II, 3 times, 5 min.
  22. Incubate with DAPI, 2 min, RT.
  23. Wash briefly with 1x PBS.
  24. Mount with mowiol.
  25. Store the slides at 4 °C for short time storage (2 weeks) or at -20 °C. It is recommended to analyze the fluorescence as soon as possible to avoid fluorophore fading.


    Figure 1. A representative figure of ImmunoFISH stained mouse hippocampus tissue. DAPI is in blue, 53BP1is in green and telomeric PNA probe is in red.

Recipes

  1. Hybridization mixture (always prepare fresh)
    Formamide
    70%
    Blocking reagent
    1x
    Tris HCl pH 7.4
    10 mM
    Telomeric PNA probe
    0.5 μM
    H2O
    to volume
  2. 10x Blocking reagent
    Prepare small aliquots and store them at -20 °C.
  3. Wash Solution I (250 ml) (always prepare fresh)
    Formamide
    175 ml
    BSA 10%
    2.5 ml
    Tris HCl 1 M pH 7.4
    2.5 ml
    H2O
    to volume
  4. Wash Solution II (350 ml) (always prepare fresh)
    Tris HCl 1 M pH 7.4
    35 ml
    NaCl 5 M
    10.5 ml
    Tween-20 10%
    2.5 ml
    H2O
    to volume

Acknowledgments

The immunofluorescence part of the protocol is adapted from Le et al. (2010). The F.d’A.d.F. laboratory is supported by FIRC (Fondazione Italiana per la Ricerca sul Cancro), AIRC (Associazione Italiana per la Ricerca sul Cancro), European Union (GENINCA, contract number 202230), HFSP (Human Frontier Science Program), AICR (Association for International Cancer Research), EMBO Young Investigator Program and Telethon.

References

  1. Fumagalli, M., Rossiello, F., Clerici, M., Barozzi, S., Cittaro, D., Kaplunov, J. M., Bucci, G., Dobreva, M., Matti, V. and Beausejour, C. M. (2012). Telomeric DNA damage is irreparable and causes persistent DNA-damage-response activation. Nat Cell Biol 14(4): 355-365.
  2. Le, O. N., Rodier, F., Fontaine, F., Coppe, J. P., Campisi, J., DeGregori, J., Laverdière, C., Kokta, V., Haddad, E. and Beauséjour, C. M. (2010). Ionizing radiation‐induced long‐term expression of senescence markers in mice is independent of p53 and immune status. Aging Cell 9(3): 398-409.

简介

该方案针对小鼠组织OCT部分的免疫FISH染色进行了优化。 它结合了DNA损伤应答因子(例如53BP1)(Le等人,2010)和针对端粒DNA的FISH的免疫荧光。

关键字:immunoFISH, DNA损伤反应, 端粒酶, Baboon组织

材料和试剂

  1. 组织
  2. OCT
  3. 4%甲醛
  4. PBS
  5. Triton
  6. 山羊血清
  7. BSA
  8. 一抗:来自Novus的53BP1 #NB 100-304兔子
  9. 第二抗体:山羊抗兔Alexa Fluor 488染料
  10. Triton
  11. 甘氨酸
  12. Mowiol 4-88试剂(Calbiochem )
  13. 甲酰胺
  14. Tris HCl,pH 7.4
  15. 端粒PNA探针(来自PANAGENE的TelC-Cy3,目录号:F1002-5)
  16. 吐温-20
  17. DAPI
  18. 杂交混合物(参见配方)
  19. 封闭试剂(Roche Diagnostics,目录号:11096176001)(参见配方)
  20. 洗涤溶液I(参见配方)
  21. 洗液II(参见配方)

设备

  1. 玻璃片
  2. 金属热块
  3. 加湿室

程序

  1. 冷冻组织置于OCT中,无固定
  2. 需要时,切成所需的厚度(8-10微米),将载玻片干燥几分钟(通常是准备其他载玻片的时间)并在-80℃下再次冷冻。
  3. 染色的日子,解冻载玻片并在4%甲醛中固定20分钟
  4. 用PBS在室温下洗涤载玻片3×5分钟
  5. 用0.5%Triton的PBS在室温下透玻片5分钟
  6. 在室温下用PBS洗涤2次,每次5分钟
  7. 在用PBS + 1%BSA稀释的5%山羊血清中封闭60分钟
  8. 在4℃下孵育:53BP1 #NB 100-304(来自Novus的兔)在PBS,2.5%山羊血清,1%BSA中1:100。 每张幻灯片使用60-80μl。
  9. 快速洗涤一次,在室温下用PBS洗涤3×10分钟
  10. 次要:在PBS + 1%BSA中的山羊抗兔(Alexa 488)(1/100)在室温下60分钟。
  11. 快速洗涤一次,用PBS RT洗3次,每次10分钟
  12. 用PFA 4%+ Triton 0.1%重新固定组织,RT 10分钟
  13. 与10mM甘氨酸在H 2 O中孵育30分钟,RT
  14. 用1×PBS清洗,3次,5分钟
  15. 准备杂交混合物,并直接30-50微升在样品上
  16. 将玻片小心地放在水滴上,不要产生气泡。
  17. 将载玻片直接放在金属加热块上,在80℃,5分钟
  18. 在加湿室中杂交,2小时,RT
  19. 从幻灯片中移除玻璃。
  20. 用洗涤溶液I洗涤两次,15分钟。
  21. 用洗涤溶液II洗涤,3次,5分钟
  22. 用DAPI孵育,2分钟,RT
  23. 用1x PBS简单冲洗。
  24. 用mowiol装载。
  25. 将载玻片存储在4°C短时储存(2周)或-20°C。 建议尽快分析荧光,以避免荧光团褪色

    图1. ImmunoFISH染色的小鼠海马组织的代表性图。 DAPI是蓝色的,53BP1是绿色的,端粒PNA探针是红色的。

食谱

  1. 杂交混合物(总是准备新鲜)
    甲酰胺
    70%
    封闭试剂
    1x
    Tris HCl pH7.4
    10 mM
    端粒PNA探针
    0.5μM
    H sub 2 O
    到卷
  2. 10x封闭试剂
    准备小份,并将其存储在-20°C
  3. 洗涤溶液I(250ml)(总是准备新鲜)
    甲酰胺
    175毫升
    BSA 10%
    2.5 ml
    Tris HCl 1M pH7.4
    2.5 ml
    H sub 2 O
    到卷
  4. 洗涤溶液II(350ml)(总是准备新鲜)
    Tris HCl 1M pH7.4
    35 ml
    NaCl 5×m/v 10.5 ml
    吐温-20 10%
    2.5 ml
    H sub 2 O
    到卷

致谢

方案的免疫荧光部分改编自Le等人(2010)。 F.d'A.d.F。实验室由FIRC(意大利人拉科尔卡),AIRC(意大利人协会),欧盟(GENINCA,合同号202230),HFSP(人类前沿科学计划),AICR(国际癌症协会研究),EMBO青年研究者计划和Telethon。

参考文献

  1. Fumagalli,M.,Rossiello,F.,Clerici,M.,Barozzi,S.,Cittaro,D.,Kaplunov,JM,Bucci,G.,Dobreva,M.,Matti,V。和Beausejour, 。 端粒DNA损伤是不可挽回的,并导致持续的DNA损伤反应激活。 Nat Cell Biol 14(4):355-365
  2. Le,O.N.,Rodier,F.,Fontaine,F.,Coppe,J.P.,Campisi,J.,DeGregori,J.,Laverdière,C.,Kokta,V.,Haddad,E.andBeauséjour, 电离辐射诱导的小鼠衰老标志物的长期表达是独立的p53和免疫状态。 老化细胞 9(3):398-409。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Rossiello, F., Fumagalli, M. and di Fagagna, F. d. (2013). ImmunoFISH for Mice and Baboons Frozen Sections. Bio-protocol 3(24): e1000. DOI: 10.21769/BioProtoc.1000.
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