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[Bio101] Immunofluorescence on Frozen Tissue Sections
[Bio101] 冷冻组织切片的荧光免疫检验   

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Abstract

Immunofluorescence is commonly used to determine the cellular or tissue localization of a protein of interest. Immunofluorescence can also be used as a qualitative measure of protein expression.

Materials and Reagents

  1. Primary antibody
  2. Alexa fluor secondary antibodies (Life Technologies, InvitrogenTM)
  3. Horse serum (Santa Cruz Biotechnology, catalog number: sc-2483 )
  4. Hoechst 33342 nuclear dye (Life Technologies, InvitrogenTM, catalog number: H1399 )
  5. ProLong gold antifade mounting reagent (Life Technologies, InvitrogenTM, catalog number: P36934 )
  6. PAP pen (Sigma-Aldrich, catalog number: Z377821 )
  7. Nail polish (Thermo Fisher Scientific, catalog number: 50-949-071 )
  8. 100% ice cold acetone
  9. Phosphate buffered saline (PBS)
  10. Formaldehyde
  11. Methanol
  12. Triton X-100

Equipment

  1. -20 °C freezer
  2. Humidified chamber

Procedure

  1. Remove 7 μm cryosections from the freezer and immediately place them into ice-cold acetone for 10 min.
    Note: The optimal fixation conditions depend upon the antigen and the primary antibody used. Formaldehyde, acetone and methanol are all common fixatives.
  2. Wash slides in PBS and aspirate around the tissue until the slide, not the tissue, is dry. Carefully, trace around the tissue with a PAP pen.
  3. Block for 1 h at room temperature (RT) with 10% horse serum in PBS with 0.05% Triton X-100.
  4. Aspirate blocking buffer and incubate sections in a humidified camber with primary antibody, diluted in 2% horse serum in PBS with 0.05% Triton X-100, for 1 h at RT. Use enough antibody solution to completely submerge the section, about 150 μl.
    Note: Follow manufactures recommendation for antibody dilution.
  5. Aspirate primary antibody and wash in 150 μl of PBS/Triton X-100 buffer 5 times.
  6. Incubate sections in a humidified chamber with corresponding secondary Alexa Fluor488/555 antibody for 1 h at RT. Dilute secondary in 2% horse serum in PBS with 0.05% Triton X-100, using a 1:300 ratio. Use enough antibody solution to completely submerge the section, about 150 μl.
  7. Aspirate secondary antibody and wash in 150 μl of PBS/Triton X-100 buffer 2 times.
  8. Wash sections in 150 μl of PBS buffer 3 times.
  9. Incubate sections for 2 min in Hoechst 33342 nuclear dye [2 mg/ml in PBS].
  10. Aspirate secondary antibody and wash in 150 μl of PBS buffer 3 times.
  11. Allow sections to dry, add ~2 drops of ProLong gold antifade reagent to each section and place a cover slip over section.
    Note: Be careful to avoid damaging the tissue by sliding the cover slip too much and do not introduce bubbles. Aspirate excess reagent.
  12. Dry sections at RT for several hours or overnight protected from light. Once the slides are completely dry, seal the edges of the cover slip with clear nail polish. Slides can be stored at -20 °C, protected from light for several weeks.

简介

免疫荧光通常用于确定感兴趣的蛋白质的细胞或组织定位。 免疫荧光也可以用作蛋白质表达的定性测量。

材料和试剂

  1. 一抗
  2. Alexa fluor第二抗体(Life Technologies,Invitrogen TM
  3. 马血清(Santa Cruz Biotechnology,目录号:sc-2483)
  4. Hoechst 33342核染料(Life Technologies,Invitrogen TM ,目录号:H1399)
  5. ProLong金抗衰老安装试剂(Life Technologies,Invitrogen TM ,目录号:P36934)
  6. PAP笔(Sigma-Aldrich,目录号:Z377821)
  7. 指甲油(Thermo Fisher Scientific,目录号:50-949-071)
  8. 100%冰冷丙酮
  9. 磷酸盐缓冲盐水(PBS)
  10. 甲醛
  11. 甲醇
  12. Triton X-100

设备

  1. -20°C冰箱
  2. 加湿室

程序

  1. 从冰箱中取出7微米冰冻切片,立即放入冰冷的丙酮中10分钟。
    注意:最佳固定条件取决于抗原和使用的一抗。甲醛,丙酮和甲醇都是常见的固定剂。
  2. 洗涤PBS中的幻灯片,并抽吸围绕组织,直到幻灯片,而不是组织,是干燥的。小心地,用PAP笔在组织周围扫描
  3. 在室温(RT)下用含有0.05%Triton X-100的PBS中的10%马血清封闭1小时
  4. 吸出阻断缓冲液,并在加湿的外倾中孵育切片,用在含有0.05%Triton X-100的PBS中的2%马血清稀释的第一抗体在室温下孵育1小时。使用足够的抗体溶液完全浸没切片,约150μl。
    注意:按照抗体稀释的制造商建议。
  5. 吸出第一抗体,并在150μlPBS/Triton X-100缓冲液中洗涤5次
  6. 孵育加湿室与相应的次级Alexa Fluor488/555抗体在室温下1小时的切片。在含有0.05%Triton X-100的PBS中的2%马血清中稀释,使用1:300的比例。使用足够的抗体 溶液完全浸没该部分,约150μl
  7. 吸出第二抗体,并在150μlPBS/Triton X-100缓冲液中洗涤2次
  8. 在150μlPBS缓冲液中洗涤切片3次
  9. 在Hoechst 33342核染料[PBS中2mg/ml]孵育切片2分钟
  10. 吸出第二抗体,并在150μlPBS缓冲液中洗涤3次
  11. 让部分干燥,在每个部分加入〜2滴ProLong金抗衰减试剂,并在部分上放置盖玻片。
    注意:小心,以避免损坏组织,滑动太多的盖玻片,不要引入气泡。 吸出过量的试剂。
  12. 干燥切片在RT下数小时或过夜避光。 一旦幻灯片完全干燥,用清洁的指甲油密封盖玻片的边缘。 载玻片可储存于-20°C,避光照射数周。
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:Reuter, J. (2011). Immunofluorescence on Frozen Tissue Sections. Bio-protocol Bio101: e163. DOI: 10.21769/BioProtoc.163;
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