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Combined in situ Hybridization/Immunohistochemistry (ISH/IH) on Free-floating Vibratome Tissue Sections
自由浮动振动切片机中组织切片的组合原位杂交/免疫组织化学法(ISH/IH)

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Abstract

In situ hybridization and immunostaining are common techniques for localizing gene expression, the mRNA and protein respectively, within tissues. Both techniques can be applied to tissue sections to achieve similar goals, but in some cases, it is necessary to use them together. For example, complement C1q is a secreted protein complex that can target the innate immune response during inflammation. Complement has been found to be elevated early and before severe neurodegeneration in several disease models. Thus, complement may serve as an important marker for disease progression and may contribute to the pathology under certain conditions. Since complement is a secreted complex, immunostaining for C1q does not necessarily reveal where compliment is produced. In situ hybridization for complement components, C1q a, b, or c mRNA, is ideal to mark complement producing cells in tissue. In situ hybridization can be coupled with cell-type-specific immunostaining for accurate identification of the cell types involved. Protein localization and mRNA localization together can reveal details as to the relationship between complement producing and complement target cells within disease tissues. Here we outline the steps for combined in situ hybridization and immunostaining on the same tissue section. The protocol outlined here has been designed for detection of complement C1q in neurons and microglia in the mouse brain.
Provided here are two approaches for combined ISH/IH. In the 1st example, in situ hybridization of C1q mRNA is performed together with fluorescent detection of Purkinje neuron cell bodies using Calbindin-D28K antibody. In the 2nd example, C1q mRNA in situ is performed together with 3,3’-diaminobenzidine (DAB) detection of microglia using CD68 antibody. Please note that modifications to the protocol may be needed for the use of distinct probes and antibodies, as well as alternate tissue-processing methods that are not specified herein. For appropriate examples of procedure results, please see images published in Lopez et al.. (2012).

Materials and Reagents

  1. Mice (to be obtained from appropriate sources and in accordance with approved animal regulations)
  2. >200 ng/ml of purified DIG-labeled RNA probe [See Stevens et al. (2007) for source of C1q RNA probe. In general, size of probe can range from 500-1,500 bp or more. Probe size alone has not appeared to affect tissue penetration or alter background. Larger probes may have enhanced signal detection.]
  3. RNaseZAP (Life Technologies, catalog number: AM9780 )
  4. RNase free water (Life Technologies, catalog number: 10977-023 )
  5. 32% paraformaldehyde solution (VWR International, catalog number: 100496-496 )
  6. 10x RNase free phosphate buffer saline (PBS) (Life Technologies, catalog number: AM9624 )
  7. 20x RNase-free SSC buffer (Life Technologies, catalog number: 15557-036 )
  8. Formamide (Sigma-Aldrich, catalog number: 47670 )
  9. Tween-20 (Sigma-Aldrich, catalog number: P1379 )
  10. tRNA (Roche Diagnostics, catalog number: 10109525001 )
  11. Salmon testis DNA (Sigma-Aldrich, catalog number: D9156 )
  12. Heparin salt (Sigma-Aldrich, catalog number: H4784 )
  13. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
  14. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9647 )
  15. Triton X-100 (Sigma-Aldrich, catalog number: X100 )
  16. Anti-Digoxigenin-AP (fab fragment) (Roche Diagnostics, catalog number: 11093274910 )
  17. NBT/BCIP ready-to-use (Roche Diagnostics, catalog number: 11697471001 )
  18. 30% hydrogen peroxide
  19. Methanol
  20. Diethyl pyrocarbonate (DEPC) (Sigma-Aldrich, catalog number: D5758 )
  21. Rat Anti-CD68 (Bio-Rad Laboratories, AbD Serotec, catalog number: MCA1957GA )
  22. Rabbit Anti-Calbindin-D28k (Sigma-Aldrich, catalog number: C2724 )
  23. Fluoromount-G (Thermo Fisher Scientific, catalog number: OB100-01 )
  24. Anti-Rabbit Alexa 488 (Life Technologies, catalog number: A21206 )
  25. Anti-Rat HRP (Jackson ImmunoResearch Laboratories, catalog number: 712035150 )
  26. SIGMA FAST DAB (Sigma-Aldrich, catalog number: D4293 )
  27. HNPP/Fast Red TR (Roche Diagnostics, catalog number: 11758888001 )
  28. NBT/BCIP tablet (Roche Diagnostics)
  29. PFA (see Recipes)
  30. 5x SSC (see Recipes)
  31. Hybe (see Recipes)
  32. Wash 1 (see Recipes)
  33. Wash 2 (see Recipes)
  34. Block (see Recipes)
  35. DEPC block (see Recipes)
  36. HPM (see Recipes)
  37. Detection buffer (see Recipes)
  38. Stop buffer (see Recipes)

Equipment

  1. Vibratome (Leica Microsystems, model: VT1200 S or other vibratome machine)
  2. 1.5 ml or a 5 ml Eppendorf tube
  3. Cold room (4 °C)
  4. Nutator (VWR International, catalog number: 15172-203 )
  5. Hybridization oven (60 °C)

Software

  1. Photoshop, Image J, etc.

Procedure

The approach for dual immunostaining of protein and RNA in situ hybridization is sequential. The method to be performed first depends on whether the immune epitope can survive the in situ hybridization conditions. The polyclonal antibody against Calbindin-D28K, a Purkinje neuron-specific marker in the cerebellum, can be applied after C1q mRNA probe in situ hybridization. Thus, the immunostaining procedure for D28K can be performed after in situ hybridization. However, the monoclonal antibody against CD68, a microglial marker, cannot. Therefore, the immunostaining procedure for CD68 needs to be performed prior to in situ hybridization. Both processes are outlined.

  1. In situ 1st, immunostaining 2nd
    1. Isolate whole brain immediately from euthanized mouse and immerse in freshly prepared PFA. Incubate overnight at 4 °C nutating [for a standard mouse brain isolation procedure please refer to Stevens et al. (2007)].
    2. Wash fixed brains twice for 5-10 min each in RNase free 5x SSC buffer to remove excess PFA.
    3. Meanwhile, clean vibratome reservoir and blade holder with RNaseZAP and rinse with RNase free water in an effort to remove any RNases; operate with new blade.
    4. Vibratome section in RNase free 5x SSC buffer, 50 µm each section.
      Note: 5x SSC can be chilled while sectioning with vibratome to reduce potential RNase activity. We have not found this to be necessary but a precaution. Also, agarose embedding of the tissue for sectioning can be performed but is not essential.
    5. Transfer vibratome sections with sterile tweezers or brush to either a 1.5 ml or a 5 ml Eppendorf tube filled with 5x SSC buffer. Size of tube and volume used for incubating and washing will depend on amount of tissue sections. A 15 ml conical may also be appropriate. For a 1.5 ml tube washing and incubating are performed using 1 ml volumes.
      Note: Allow about 90% of total volume of liquid to be transferred between washes. For a 1.5 ml tube that would mean that free-floating tissue sections should fall and accumulate below the 100 μl mark so that 900 μl can be easily pipetted off and replaced. This approach should be adapted to greater volume tubes if used.
    6. Replace 50% of the 5x SSC buffer with Hybe and nutate for 5 min. Then allow sections to settle.
    7. Replace 90% of the supernatant with Hybe and nutate at 60 °C for ~10 min or more, until ready to add probe.
    8. Meanwhile, add 200 ng of purified DIG-labeled RNA probe per ml of Hybe to be used. Heat to 80 °C for 5 min to denature RNA probe into Hybe.
    9. Remove as much of the hybridization solution from the samples as possible and replace with Hybe with probe from step A8.
      Note: To conserve on probe and Hybe solution, samples can be incubated standing, without mixing, rotating or nutating, in the least amount of liquid needed to cover the sample, e.g. 100 μl if a 1.5 ml Eppendorf tube is used.
    10. Hybridize at 60 °C for at least 16 h nutating.
      Note: Again, if using low volumes of Hybe w/probe to cover tissue sections, mixing while hybridizing is not necessary. The probe should diffuse adequately at high temperature and during long 16 h or more incubation periods.
    11. After hybridization, replace or dilute hybridization solution on samples with Hybe without probe and incubate at 60 °C for 15 min nutating.
      Note: Hybe and wash 1 and 2 buffers are usually prewarmed to 60 °C, but protocol has been successfully performed without heating the buffers for this and the following wash steps.
    12. Wash 2x with wash 1 at 60 °C for 15 min nutating.
    13. Wash 2x with wash 2 at 60 °C for 15 min nutating.
    14. Replace wash 2 with block and incubate at room temp for 10 min.
    15. Replace block again to dilute wash 2 further.
    16. Incubate hybridized sections at 4 °C overnight in fresh block containing 1: 1,000 dilution of anti-Digoxigenin-AP, Fab fragments and Rabbit anti-Calbindin-D28K.
    17. Wash 4x with block at room temp, >10 min each wash.
    18. Incubate sections at 4 °C overnight in block containing 1: 1,000 dilution of anti-rabbit Alexa488 secondary, or other appropriate fluorescent secondary (available from Jackson Immunoresearch or Life Technologies).
      Note: Anti-Digoxigenin-AP, Fab fragments at 1: 1,000 can be added again if fear of losing signal from overnight wash.
    19. Wash 4x with block at room temp, at least 10 min each wash.
    20. Gradually replace block with detection buffer, a 50% exchange followed by 90% exchange of liquid in tube and then a subsequent 90% exchange.
    21. Dissolve a ready-to-use NBT/BCIP tablet in 10 ml of dH2O.
    22. Exchange detection buffer with NBT/BCIP solution and incubate in the dark without motion.
    23. Samples can be transferred from tubes to dishes for better color monitoring.
      Note: Purple precipitate can form in a few minutes, hours or left overnight at 4 °C depending on probe and expression levels of target.
    24. Once color spots in tissue sections are detected, replace NBT/BCIP solution with stop buffer.
    25. After development mount sections on slides and coverslip with Fluoromount-G.
    26. mRNA in situ can be visualized with bright field or phase and immunostaining, Calbindin D28K can be seen by fluorescent microscopy.
    27. The images can then be overlayed using software: Photoshop, Image J, etc.
      Note: Alternatively, NBT/BCIP colormetric reaction can be replaced with a fluorescent substrate such as HNPP/Fast Red TR for double fluorescence detection.

  2. Immunostaining 1st, in situ 2nd
    1. Isolate whole brain immediately from euthanized mouse and immerse in freshly prepared PFA. Incubate overnight at 4 °C nutating.
    2. Wash fixed brains twice for 5-10 min each in RNase free PBS to remove excess PFA.
    3. Meanwhile, clean vibratome reservoir and blade holder with RNaseZAP and rinse with RNase free water in an effort to remove any RNases; operate with new blade.
    4. Vibratome section in RNase free PBS buffer, 50 µm each section.
    5. Transfer vibratome sections to either a 1.5 ml or a 5 ml Eppendorf tube, depending on amount of tissue sections used, and fill with PBS.
    6. Replace 50% of PBS with HPM (hydrogen peroxide in methanol to quench endogenous peroxidase activity) and mix.
    7. Replace as much of the mix with HPM.
    8. Incubate at room temp for 30 min nutating.
    9. Replace HPM gradually with RNase free PBS with a series of 25%, 50%, 100% volume exchanges for 1-2 min each.
    10. Replace PBS with DEPC block at room temp.
    11. Incubate for 10 min then replace with fresh DEPC block.
    12. Incubate free-floating sections at 4 °C overnight in DEPC block containing 1: 1,000 dilution of rat anti-CD68.
    13. Wash 4x with DEPC block at room temp, at least 10 min each wash.
    14. Wash 2x with RNase free PBS to remove DEPC block, ~2-5min each wash.
    15. Fix with PFA for 30 min-1 h at room temp.
    16. Wash sections for 5 min, 2x in RNase free 5x SSC buffer to remove excess PFA.
    17. Follow steps A6-15 above.
    18. Incubate hybridized sections at 4 °C overnight in block containing 1: 1,000 dilution of anti-Digoxigenin-AP, Fab fragments and 1: 10,000 dilution of anti-rat HRP secondary.
    19. Wash 4x with block at room temp, at least 10 min each wash.
    20. Follow steps A20-24 above.
    21. After developing sections with NBT/BCIP reaction switch to PBS then develop DAB reaction according to Sigma protocol for FAST DAB reaction.
      Note: Do not use metal enhancer, as the purple-blue from the NBT/BCIP reaction overlayed with the brown produced from the DAB reaction can produce a blackish color that can be masked by metal enhancement of DAB reaction, which yields a blue-black color. If NBT/BCIP reaction is expected to take a long time to develop, the DAB reaction may be performed first. Optimum conditions would have to be worked out for each experiment.
    22. After development sections were mounted on slides with Fluoromount-G and imaged with light microscopy. Alternatively, for double fluorescence microscopy applications, HNPP/Fast Red TR for AP reaction and Tyramide signal amplification (TSA) with Alexa Fluor dyes for HRP reaction can be used.

Representative data



Figure 1. Example result from procedure B. Immunostaining 1st, in situ 2nd.
Image shows Purkinje neuron layer (cerebellar Purkinje neurons are identified by morphology) of a neurodegenerative disease mouse with anti-CD68 DAB staining (brown) and C1q mRNA NBT/BCIP staining (purple). In this image the C1q staining was underdeveloped and appears weak in order to demonstrate the distinct brown DAB staining. Images were taken using a Zeiss Axioplan II microscope.

For additional representative data please see figures from the following Reference (Lopez et al., 2012). 

Notes

  1. DEPC is a carcinogenic and should be handles with care. As an alternative to DEPC, an RNase inhibitor can be substituted. Some publications have described the use of 5 mg/ml sodium heparin to serve as an RNase inhibitor. See Cockwell et al. (1998).

Recipes

  1. PFA
    1x PBS
    4% paraformaldehyde
  2. 5x SSC
    Dilute 20x SSC with RNAse free dH2O: 1 vol. 20x SSC plus 3 vol. dH2O
  3. Hybe
    50% formamide
    5x SSC
    0.1% Tween-20
    0.5% SDS
    500 μg/ml tRNA
    500 μg/ml salmon sperm DNA
    50 μg/ml heparin salt
  4. Wash 1
    5x SSC
    0.1% Tween-20
  5. Wash 2
    0.2x SSC
    0.1% Tween-20
  6. Block
    1x PBS
    2% BSA
    0.2% Triton X-100
  7. DEPC block
    1x PBS
    2% BSA
    0.2% Triton X-100
    0.01% DEPC
    Caution: DEPC is toxic, use protection and proper disposal techniques.
  8. HPM
    Methanol
    0.6%-1% hydrogen peroxide
    0.01% Tween-20
  9. Detection buffer
    0.1 M Tris-HCl (pH 9.5)
    0.1 M NaCl
    0.01% Tween-20
  10. Stop buffer
    10 mM Tris-HCl (pH 8.0)
    1 mM EDTA
    0.01% Tween-20

Acknowledgments

This protocol has been adapted from Lopez et al. (2012).

References

  1. Cockwell, P., Howie, A. J., Adu, D. and Savage, C. O. (1998). In situ analysis of C-C chemokine mRNA in human glomerulonephritis. Kidney Int 54(3): 827-836.
  2. Lopez, M. E., Klein, A. D. and Scott, M. P. (2012). Complement is dispensable for neurodegeneration in Niemann-Pick disease type C. J Neuroinflammation 9(1): 1-8.
  3. Navone, S. E., Marfia, G., Invernici, G., Cristini, S., Nava, S., Balbi, S., Sangiorgi, S., Ciusani, E., Bosutti, A., Alessandri, G., Slevin, M. and Parati, E. A. (2013). Isolation and expansion of human and mouse brain microvascular endothelial cells. Nat Protoc 8(9): 1680-1693.
  4. Stevens, B., Allen, N. J., Vazquez, L. E., Howell, G. R., Christopherson, K. S., Nouri, N., Micheva, K. D., Mehalow, A. K., Huberman, A. D., Stafford, B., Sher, A., Litke, A. M., Lambris, J. D., Smith, S. J., John, S. W. and Barres, B. A. (2007). The classical complement cascade mediates CNS synapse elimination. Cell 131(6): 1164-1178.

简介

原位杂交和免疫染色是分别定位组织内基因表达,mRNA和蛋白质的常用技术。两种技术可以应用于组织切片以实现类似的目标,但是在一些情况下,有必要一起使用它们。例如,补体C1q是可以靶向炎症期间的先天免疫应答的分泌的蛋白复合物。已经发现补体在几种疾病模型中早期升高和严重神经变性之前升高。因此,补体可以作为疾病进展的重要标志物,并且可能有助于在某些条件下的病理学。由于补体是分泌的复合物,C1q的免疫染色不一定揭示产生补偿的位置。补体成分,C1q a,b或c mRNA的原位杂交是理想的标记组织中产生补体的细胞。原位杂交可以与细胞类型特异性免疫染色偶联以准确鉴定所涉及的细胞类型。蛋白质定位和mRNA定位一起可以揭示关于疾病组织内补体产生和补体靶细胞之间的关系的细节。在这里我们概述在同一组织切片上的组合原位杂交和免疫染色的步骤。这里概述的协议已设计用于检测小鼠脑神经元和小胶质细胞中的补体C1q。
这里提供了两种组合ISH/IH的方法。在第一个实施例中,C1q mRNA的原位杂交与使用Calbindin-D28K抗体的Purkinje神经元细胞体的荧光检测一起进行。在第二个实施例中,使用CD68抗体与小胶质细胞的3,3'-二氨基联苯胺(DAB)一起进行C1q mRNA原位。请注意,对于使用不同的探针和抗体以及本文未指定的替代组织处理方法,可能需要对方案进行修改。有关程序结果的适当示例,请参阅Lopez等人发表的图像。。 (2012)。

材料和试剂

  1. 小鼠(从适当的来源获得,并根据批准的动物规章)
  2. 200ng/ml纯化的DIG-标记的RNA探针[参见Stevens等人(2007))用于C1q RNA探针的来源。 通常,探针的大小可以在500-1,500bp或更大的范围内。 单独的探针大小似乎没有影响组织穿透或改变背景。 较大的探头可能具有增强的信号检测。]
  3. RNaseZAP(Life Technologies,目录号:AM9780)
  4. 无RNA酶水(Life Technologies,目录号:10977-023)
  5. 32%多聚甲醛溶液(VWR International,目录号:100496-496)
  6. 10x无RNA酶的磷酸盐缓冲盐水(PBS)(Life Technologies,目录号:AM9624)
  7. 20x无RNase的SSC缓冲液(Life Technologies,目录号:15557-036)
  8. 甲酰胺(Sigma-Aldrich,目录号:47670)
  9. Tween-20(Sigma-Aldrich,目录号:P1379)
  10. tRNA(Roche Diagnostics,目录号:10109525001)
  11. 鲑鱼睾丸DNA(Sigma-Aldrich,目录号:D9156)
  12. 肝素盐(Sigma-Aldrich,目录号:H4784)
  13. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L3771)
  14. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A9647)
  15. Triton X-100(Sigma-Aldrich,目录号:X100)
  16. 抗洋地黄毒苷-AP(fab片段)(Roche Diagnostics,目录号:11093274910)
  17. NBT/BCIP即用型(Roche Diagnostics,目录号:11697471001)
  18. 30%过氧化氢
  19. 甲醇
  20. 焦碳酸二乙酯(DEPC)(Sigma-Aldrich,目录号:D5758)
  21. 大鼠抗CD68(Bio-Rad Laboratories,AbD Serotec,目录号:MCA1957GA)
  22. 兔抗钙结合蛋白-D28k(Sigma-Aldrich,目录号:C2724)
  23. Fluoromount-G(Thermo Fisher Scientific,目录号:OB100-01)
  24. 抗兔Alexa 488(Life Technologies,目录号:A21206)
  25. 抗大鼠HRP(Jackson ImmunoResearch Laboratories,目录号:712035150)
  26. SIGMA FAST DAB(Sigma-Aldrich,目录号:D4293)
  27. HNPP/Fast Red TR(Roche Diagnostics,目录号:11758888001)
  28. NBT/BCIP片剂(Roche Diagnostics)
  29. PFA(请参阅食谱)
  30. 5x SSC(参见配方)
  31. Hybe(见食谱)
  32. 清洗1(参见配方)
  33. 洗2(见配方)
  34. 块(参见配方)
  35. DEPC块(参见配方)
  36. HPM(参见配方)
  37. 检测缓冲区(参见配方)
  38. 停止缓冲液(参见配方)

设备

  1. Vibratome(Leica Microsystems,型号:VT1200 S或其他vibratome机器)
  2. 1.5ml或5ml Eppendorf管
  3. 冷室(4℃)
  4. Nutator(VWR International,目录号:15172-203)
  5. 杂交炉(60℃)

软件

  1. Photoshop,Image J,等。

程序

用于蛋白质和RNA的原位杂交的双重免疫染色的方法是连续的。首先要执行的方法取决于免疫表位是否能够在原位杂交条件下存活。针对小脑中Purkinje神经元特异性标记的Calbindin-D28K的多克隆抗体可以在C1q mRNA探针原位杂交后应用。因此,D28K的免疫染色程序可以在原位杂交后进行。然而,抗CD68的单克隆抗体(一种小胶质细胞标志物)不能。因此,CD68的免疫染色程序需要在原位杂交之前进行。概述了这两个过程。

  1. 原位 1 st ,免疫染色2 nd
    1. 立即分离整个大鼠从安乐死的小鼠,并沉浸在新鲜制备的PFA。在4℃章动孵育过夜[对于标准小鼠脑分离程序,请参考Stevens等人(2007)]。
    2. 在无RNA酶的5×SSC缓冲液中洗涤固定的脑两次,每次5-10分钟,以除去过量的PFA
    3. 同时,用RNaseZAP清洁vibratome储存器和刀片夹,并用RNase自由水冲洗,以去除任何RNA酶;使用新刀片
    4. Vibratome部分在RNase游离5x SSC缓冲液,每个部分50μm。
      注意:5x SSC可以在用vibratome切片时冷冻,以降低潜在的RNase活性。我们没有发现这是必要的,但是预防措施。此外,可以进行用于切片的组织的琼脂糖包埋,但不是必需的。
    5. 转移vibratome部分用无菌镊子或刷到充满5x SSC缓冲液的1.5 ml或5 ml Eppendorf管。用于孵育和洗涤的管和容积的大小将取决于组织切片的量。 15ml锥形也可以是合适的。对于1.5ml管,使用1ml体积进行洗涤和孵育 注意:允许在洗液之间转移总体积的约90%的液体。对于1.5ml管,这意味着自由浮动的组织切片应该下降并积累在100μl标记以下,以便900μl可以轻易吸取和替换。此方法应适用于体积较大的管(如果使用)。
    6. 用Hybe和nutate替换50%的5×SSC缓冲液5分钟。然后允许部分结算。
    7. 用Hybe和nutate在60℃下将90%的上清液更换〜10分钟或更长时间,直到准备添加探针。
    8. 同时,添加200ng纯化的DIG标记的RNA探针/ml Hybe使用。加热至80℃5分钟以使RNA探针变性至Hybe。
    9. 从样品中尽可能多地去除杂交溶液,并用来自步骤A8的探针替换Hybe 注意:为了保存在探针和Hybe溶液上,可以将样品静置,不混合,旋转或章动,在覆盖样品所需的最少量的液体中孵育,例如。如果使用1.5ml Eppendorf管,则为100μl。
    10. 在60℃下杂交至少16小时章动。
      注意:同样,如果使用低体积的Hybe w /探针覆盖组织切片,则不必进行杂交混合。探针应在高温和长达16小时或更长的孵育期内充分扩散。
    11. 杂交后,用无探针的Hybe替换或稀释样品上的杂交溶液,并在60℃下孵育15分钟。 注意:Hybe和Wash 1和2缓冲液通常预热至60°C,但是已经成功地进行了方案,不需要为此和以下清洗步骤加热缓冲液。
    12. 用60℃的洗涤1洗涤2次,15分钟章动
    13. 用60℃的洗涤2洗涤2次,15分钟章动
    14. 将块2替换为块,并在室温下孵育10分钟
    15. 再次更换块以进一步稀释洗液2
    16. 在4℃下在含有抗洋地黄毒苷-AP,Fab片段和兔抗钙结合蛋白-D28K的1:1000稀释液的新鲜块中孵育杂交切片。
    17. 在室温下用块洗涤4次,每次洗涤> 10分钟
    18. 在4℃下在含有1:1000稀释的抗兔Alexa488二抗或其他合适的荧光二抗(可从Jackson Immunoresearch或Life Technologies获得)的块中孵育切片过夜。
      注意:如果担心从过夜洗涤中失去信号,可以再次加入1:1,000的抗洋地黄毒苷-AP,Fab片段。
    19. 室温下冲洗4次,每次冲洗至少10分钟。
    20. 逐渐用检测缓冲液替换块,50%交换,随后在管中90%交换液体,然后随后90%交换。
    21. 将即用的NBT/BCIP片剂溶解在10ml dH 2 O中。
    22. 用NBT/BCIP溶液交换检测缓冲液,在黑暗中孵育,无运动
    23. 样品可以从试管转移到培养皿中以获得更好的颜色监测 注意:根据探针和目标的表达水平,紫色沉淀可在几分钟,几小时内形成或在4℃过夜。
    24. 一旦检测到组织切片中的色斑,用停止缓冲液替代NBT/BCIP溶液。
    25. 开发后载玻片上的部分和盖玻片与Fluoromount-G。
    26. mRNA原位可通过明视野或相和免疫染色显现,可通过荧光显微镜观察到Calbindin D28K。
    27. 然后可以使用软件:Photoshop,Image J,等覆盖图像。
      注意:或者,NBT/BCIP比色反应可以用荧光底物例如HNPP/Fast Red TR代替双荧光检测。

  2. 免疫染色1 st , 2 nd
    1. 立即分离整个大鼠从安乐死的小鼠,并沉浸在新鲜制备的PFA。 在4℃章动孵育过夜。
    2. 在无RNase的PBS中洗涤固定的脑两次,每次5-10分钟,以除去过量的PFA
    3. 同时,用RNaseZAP清洁vibratome储存器和刀片夹,并用RNase自由水冲洗,以去除任何RNA酶; 使用新刀片
    4. Vibratome切片在无RNA酶的PBS缓冲液中,每个切片50μm
    5. 根据所使用的组织切片数量,将振荡器切片转移到1.5 ml或5 ml Eppendorf管中,并填充PBS。
    6. 用HPM(甲醇中的过氧化氢,淬灭内源性过氧化物酶活性)替换50%的PBS,混匀
    7. 用HPM替换尽可能多的混合物。
    8. 在室温下孵育30分钟章动
    9. 用无RNase的PBS逐步替换HPM,每次用25%,50%,100%体积交换,每次1-2分钟。
    10. 在室温下用DEPC块替换PBS。
    11. 孵育10分钟,然后更换新鲜的DEPC块。
    12. 在4℃下,将自由漂浮的切片在含有1:1000稀释的大鼠抗CD68的DEPC块中孵育过夜。
    13. 在室温下用DEPC块洗涤4次,每次洗涤至少10分钟
    14. 用无RNase的PBS洗涤2次以去除DEPC阻滞,每次洗涤〜2-5min
    15. 在室温下用PFA固定30分钟-1小时。
    16. 在无RNA酶的5×SSC缓冲液中洗涤切片5分钟,2次,以除去过量的PFA
    17. 按照上述步骤A6-15。
    18. 孵育杂交切片在4℃下过夜,包含1:1000稀释的抗洋地黄毒苷-AP,Fab片段和1:10,000稀释的抗大鼠HRP二抗。
    19. 在室温下用块洗涤4次,每次洗涤至少10分钟。
    20. 按照上述步骤A20-24。
    21. 在用NBT/BCIP反应切开显影切片后,然后根据用于FAST DAB反应的Sigma方案开发DAB反应。
      注意:不要使用金属增强剂,因为来自NBT/BCIP反应的紫色蓝色覆盖有从DAB反应产生的棕色,可以产生黑色的颜色,其可以被DAB反应的金属增强掩蔽,这产生了蓝黑色。如果预期NBT/BCIP反应需要很长时间才能形成,则可以首先进行DAB反应。最佳条件必须为每个实验确定。
    22. 显影后,将切片用Fluoromount-G安装在载玻片上,并用光学显微镜成像。或者,对于双荧光显微镜应用,可以使用用于AP反应的HNPP/Fast Red TR和用于HRP反应的用Alexa Fluor染料的酪胺信号放大(TSA)。

代表数据



图1.来自方法B的实施例结果。免疫染色1 2 。图像显示Purkinje神经元层Purkinje神经元通过形态学识别神经变性 用抗CD68 DAB染色(棕色)和C1q mRNA NBT/BCIP染色(紫色)染色。 在该图像中,C1q染色不发达,并且看起来很弱,以便证明不同的棕色DAB染色。 使用Zeiss Axioplan II显微镜拍摄图像
有关其他代表性数据,请参阅以下参考文献(Lopez等人,2012年)的数据。

笔记

  1. DEPC是致癌物,应小心处理。 作为DEPC的替代物,可以用RNase抑制剂代替。 一些出版物已经描述了使用5mg/ml肝素钠作为RNA酶抑制剂。 参见Cockwell等人 (1998)。

食谱

  1. PFA
    1x PBS
    4%多聚甲醛
  2. 5x SSC
    用无RNA酶的dH 2 O稀释20×SSC:1体积。 20x SSC + 3vol。 dH 2 2 O
  3. Hybe
    50%甲酰胺 5x SSC
    0.1%Tween-20
    0.5%SDS
    500μg/ml tRNA
    500μg/ml鲑鱼精子DNA 50μg/ml肝素盐
  4. 洗涤1
    5x SSC
    0.1%Tween-20
  5. 清洗2
    0.2x SSC
    0.1%Tween-20
  6. 阻止
    1x PBS
    2%BSA
    0.2%Triton X-100
  7. DEPC块
    1x PBS
    2%BSA
    0.2%Triton X-100 0.01%DEPC
    警告:DEPC有毒,使用保护和适当的处置技术。
  8. HPM
    甲醇
    0.6%-1%的过氧化氢 0.01%Tween-20
  9. 检测缓冲区
    0.1M Tris-HCl(pH9.5)
    0.1 M NaCl
    0.01%Tween-20
  10. 停止缓冲区
    10mM Tris-HCl(pH8.0) 1mM EDTA
    0.01%Tween-20

致谢

该协议已经从Lopez等人修改(2012)。

参考文献

  1. Cockwell,P.,Howie,A.J.,Adu,D。和Savage,C.O.(1998)。 对人类肾小球肾炎中CC趋化因子mRNA的原位分析。 Kidney Int 54(3):827-836。
  2. Lopez,M.E.,Klein,A.D.and Scott,M.P。(2012)。 补体对于C型尼曼 - 皮克病的神经退行性疾病是不必要的。 J Neuroinflammation 9(1):1-8。
  3. Navone,SE,Marfia,G.,Invernici,G.,Cristini,S.,Nava,S.,Balbi,S.,Sangiorgi,S.,Ciusani,E.,Bosutti,A.,Alessandri, ,M。和Parati,EA(2013)。 人和小鼠脑微血管内皮细胞的分离和扩增 Nat Protoc 8(9):1680-1693。
  4. Stevens,B.,Allen,NJ,Vazquez,LE,Howell,GR,Christopherson,KS,Nouri,N.,Micheva,KD,Mehalow,AK,Huberman,AD,Stafford, AM,Lambris,JD,Smith,SJ,John,SWand Barres,BA(2007)。 经典补体级联介导CNS突触消除。 细胞 131(6):1164-1178。
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引用:Lopez, M. E. (2014). Combined in situ Hybridization/Immunohistochemistry (ISH/IH) on Free-floating Vibratome Tissue Sections. Bio-protocol 4(18): e1243. DOI: 10.21769/BioProtoc.1243.
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