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This protocol describes a method for efficient immunolabelling of thin tissue slices containing a few rows of intact intestinal crypts, which yields large numbers of them being oriented favorably for recording stacks of optical sections aligned with the crypt long axis (Bellis et al., 2012). The latter can then be used for cell positional analysis, 3D-reconstruction and -analysis. The simple epithelium lining the small intestine is organized into contiguous crypts of Lieberkühn (Potten, 1998; Barker et al., 2012; De Mey and Freund, 2013) several of which making up a crypt/villus unit. Each crypt is a multicellular proliferation unit with a tight hierarchical organization. Under steady state conditions, the epithelium is continuously and rapidly renewed, driven by divisions of multipotent intestinal SCs near the crypt base and cell removal from the villus tip. Techniques for analyzing the organization of the crypts play an important role in the field. Maximal efficiency is obtained by using optical sections obtained from confocal scanning and/or Nomarski optics passing through the center of the longitudinal crypt axis to view the crypt as two cell columns of hierarchical lineage starting from cells positioned at or near the crypt base. This enables positional analysis of certain cellular capacities like performing DNA synthesis, undergoing mitosis and apoptosis (Caldwell et al., 2007; Fleming et al., 2007; Quyn et al., 2010), responding to injury (Potten et al., 1997), or expressing genes (Barker et al., 2012; Bjerknes et al., 2012; Itzkovitz et al., 2012). Our protocol has allowed us to demonstrate that some divisions are asymmetric with respect to cell fate and the occurrence of oriented cell division (OCD) in 80% of the proliferating cells in the upper stem cell and transit amplifying zones. It has further revealed planar cell polarities which are important for crypt homeostasis and stem cell biology and alterations in apparently normal crypts and microadenomas of mice carrying germline Apc mutations shedding new light on the first stages of progression towards colorectal cancer.

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Immunolabelling of Thin Slices of Mouse Descending Colon and Jejunum
小鼠降结肠和空肠薄切片的免疫标记

干细胞 > 成体干细胞 > 肠道干细胞
作者: Julien Bellis
Julien BellisAffiliation: Laboratory of Biophotonics and Pharmacology, CNRS UMR 7213, University of Strasbourg, Illkirch, France
Bio-protocol author page: a920
Isabelle Duluc
Isabelle DulucAffiliation: INSERM UMR S1113, University of Strasbourg, Strasbourg, France
Bio-protocol author page: a921
Jean-Noël Freund
Jean-Noël FreundAffiliation: INSERM UMR S1113, University of Strasbourg, Strasbourg, France
Bio-protocol author page: a922
 and Jan R. De Mey
Jan R. De MeyAffiliation: Laboratory of Biophotonics and Pharmacology, CNRS UMR 7213, University of Strasbourg, Illkirch, France
For correspondence: jan.de-mey@unistra.fr
Bio-protocol author page: a923
Vol 3, Iss 20, 10/20/2013, 3584 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.942

[Abstract] This protocol describes a method for efficient immunolabelling of thin tissue slices containing a few rows of intact intestinal crypts, which yields large numbers of them being oriented favorably for recording stacks of optical sections aligned with the crypt long axis (Bellis et al., 2012). The latter can then be used for cell positional analysis, 3D-reconstruction and -analysis. The simple epithelium lining the small intestine is organized into contiguous crypts of Lieberkühn (Potten, 1998; Barker et al., 2012; De Mey and Freund, 2013) several of which making up a crypt/villus unit. Each crypt is a multicellular proliferation unit with a tight hierarchical organization. Under steady state conditions, the epithelium is continuously and rapidly renewed, driven by divisions of multipotent intestinal SCs near the crypt base and cell removal from the villus tip. Techniques for analyzing the organization of the crypts play an important role in the field. Maximal efficiency is obtained by using optical sections obtained from confocal scanning and/or Nomarski optics passing through the center of the longitudinal crypt axis to view the crypt as two cell columns of hierarchical lineage starting from cells positioned at or near the crypt base. This enables positional analysis of certain cellular capacities like performing DNA synthesis, undergoing mitosis and apoptosis (Caldwell et al., 2007; Fleming et al., 2007; Quyn et al., 2010), responding to injury (Potten et al., 1997), or expressing genes (Barker et al., 2012; Bjerknes et al., 2012; Itzkovitz et al., 2012). Our protocol has allowed us to demonstrate that some divisions are asymmetric with respect to cell fate and the occurrence of oriented cell division (OCD) in 80% of the proliferating cells in the upper stem cell and transit amplifying zones. It has further revealed planar cell polarities which are important for crypt homeostasis and stem cell biology and alterations in apparently normal crypts and microadenomas of mice carrying germline Apc mutations shedding new light on the first stages of progression towards colorectal cancer.

[Abstract]

Materials and Reagents

  1. Mouse
  2. Isoflurane gas (Abbott Laboratories)
  3. Dissection pan wax (black) (Fisher Scientific, catalog number: S17432 )
  4. Pipes
  5. HEPES
  6. EGTA
  7. MgSO4
  8. Triton-X-100
  9. Taxol (Paclitaxel from Sigma-Aldrich, catalog number: T7191 )
  10. PBS
  11. NaN3
  12. Sodium deoxycholate
  13. BSA 10% in PBS
  14. Tri-sodium citrate dihydrate (Merck KGaA, catalog number: 567446 )
  15. 1 N HCl (1 mol/L) (Merck KGaA, catalog number: 1090571000 )
  16. Paraformaldehyde (PFA) 16% solution, EM grade (Electron Microscopy Sciences, catalog number: 15710 )
  17. Phalloidin-Alexa 568 (Life Technologies, Molecular Probes®, catalog number: 12380 )
    Note: The Alexa type may be chosen according to available other fluorophores.
  18. DAPI: 1 mg/ml stock solution in PBS (stored at 4 °C)
  19. Alexa-labeled secondary antibodies. In our study, these were from Life Technologies- Molecular Probes goats anti (species) IgG (H + L) and all highly cross-adsorbed to assure absence of cross-species-reactivity.
    Note: For any combination of primary antibodies, a choice of corresponding secondary antibodies and fluorophore wavelengths has to be made. When as in our case, Phalloidin-Alexa 568 (red fluorescence) is used for marking microfilaments made of actin, and DAPI for DNA, secondary antibodies are usually labelled with Alexa 488 (green fluorescence) and Alexa 647 (far-red fluorescence). The latter is recommended for antigens giving weak signals, since it is very well detected by current confocal microscopy systems. When Phalloidin could not be used, Alexa 568 or 555 (red fluorescence) labelled secondary antibodies could be used in addition.
    Note: In our study, no commercial primary antibodies were used.
  20. Prolong Gold mounting medium without DAPI (Life Technologies, catalog number: P36934 )
  21. 2x PHEM (see Recipes)
  22. Fixative (see Recipes)
  23. 20 mM Sodium citrate buffer (see Recipes)
  24. Solutions of primary affinity-purified antibodies (see Recipes)
  25. Solutions of secondary antibodies (see Recipes)

Equipment

  1. Thermostatic water bath
  2. Tem Sega evaporator apparatus for isoflurane anesthesia
  3. Scissors 3 cm and microscissors (Fine Science Tools)
  4. Forceps n°5 (Fine Science Tools)
  5. Binocular
  6. 30 G 1/2” needles
  7. U-100 Insulin syringe + needle
  8. Microscope slides 76 x 26 x 1.1 mm
  9. 1.5 ml Eppendorf tubes
  10. Micropipettes
  11. 18 x 18 mm glass coverslips Nr. 1
  12. Fast confocal microscope (we use a Leica SP5 confocal microscope)
  13. 63x NA 1.4 oil immersion lens

Procedure

  1. Sample preparation
    1. The mouse needs to be anesthetized during tissue collection. 10 min before anesthetizing it, prepare 15 ml PHEM 1x (from PHEM 2x) in a 50 ml plastic tube and warm it to 37 °C in a thermostatic water bath. Transport the tube in a recipient filled with water at 37 °C to the dissection room and use within minutes.
    2. Anesthetize the mouse using isoflurane inhalation with the help of the Tem Sega evaporator.
    3. While continuing anesthetizing, place it under a binocular microscope placed in a chemical extraction hood, so that the operator does not inhale isoflurane and formaldehyde gases.
    4. Open the abdomen with fine scissors, localize the distal (descending) colon with respect to the anus, free it from surrounding tissue and transect it about 2.5 cm from the latter. For the jejunum, transect it about 2.5 cm from the caecum/proximal colon. For a scheme of the mouse digestive tract, see: http://www.informatics.jax.org/cookbook/figures/figure76.shtml.
      Note: For our study, preservation of sensitive cytoskeleton-based structures like mitotic spindles was essential and could only be achieved by rapid handling. We therefore used one animal per intestinal segment and did not straighten out the intestine. For other studies, for example the presence of certain transcription factors in the nucleus of certain cell types, this is probably less critical.
    5. Using a syringe, flush the colon or jejunum section with PHEM 1x at 37 °C.
      Note: 37 °C is important for avoiding changes to mitotic spindles that are temperature sensitive. For other studies, this may be less critical.
    6. Flush it immediately with fixative supplemented with 15 μM taxol.
      Note: The fixative contains PHEM buffer, known to optimize the preservation of Ca2+ sensitive microtubules (Schliwa and van Blerkom, 1981) and some Triton-X-100 to accelerate the speed of fixation. It is supplemented with taxol, to further prevent microtubule loss. Taxol indeed binds to microtubules and makes them resistant to degradation by formaldehyde fixation. Without added taxol, spindles were found to be shorter and sometimes distorted, making the measurement of spindle angles as described in our study unreliable. When preservation of microtubules is not essential, taxol may be left out. PHEM buffer, however, is recommended since it prevents pH changes during formaldehyde fixation better then for example PBS.
    7. Cut out a segment of 1-2 cm and place it in a cup filled with hardened dissection pan wax (black) filled with 5 ml of fixative at room temperature. Open the colon or jejunum segments with fine scissors by cutting them along their length.
    8. Sacrifice the mouse by cutting through the septum into the heart with scissors.
    9. Pin the tissue flattened and lightly stretched on the wax surface, mucosa up, and continue fixation for 40 min at room temperature.
    10. After about ten minutes, while in fixative, cut the tissue into small 1.5 mm3 cubes, with the help of microscissors.
    11. Transfer these into a 1.5 ml Eppendorf tube and after a total of 40 minutes rinse them three times 10 min in PBS. They can now be stored at 4 °C in PBS supplemented with 8 mM NaN3 for several weeks.
    12. Using a cut-off blue tip on a micropipette, transfer a few pieces onto a microscope slide placed under a binocular. For colon fragments only, remove the muscle lining using two thin 30 G 1/2” needles fixed on a U-100 insulin syringe. To this end, the muscle lining must first be positioned underneath the mucosa layer. They are then separated by inserting one needle between the layers and using it for keeping the muscle layer fixed in place, while using the other to slide or peel the mucosa away.
      Note: The muscle lining of the jejunum is thin and fragile, and does not need to be removed.
    13. Using one of the needles as a cutting device while holding in place the mucosa fragment with the other one cut away thin around 1.5 mm long slices containing two to three rows of contiguous crypts. Transfer 30-40 such slices into a 1.5 ml Eppendorf tube. All subsequent steps are performed in such Eppendorf tubes, one per primary antibody combination.
    14. Mount the tubes with about 30 mucosa slices on a rotating wheel and incubate successively during 30 min with 1 ml of PBS containing 200 mM NH4Cl, 3% sodium deoxycholate in H2O, 0.5% Triton-X-100 in PBS and PBS/BSA 1%/Triton 0.2% for blocking. Use a 1 ml blue tip mounted on a micropipette to remove liquids after each incubation step after the slices have sunk by gravity to the tube bottom. Take care to avoid losing slices by holding the tube against a light source.
      Note: Preparation of the deoxycholate solution in H2O is imperative.
    15. For labeling with certain antibodies (for example against the transcription factors Atoh1 or Cdx2), perform antigen retrieval by incubating the tubes containing the samples with 1 ml of 0.1 mM sodium citrate buffer, pH 6.0 (prepared from a 20x stock buffer) placed in a block heater at 95 °C for 30 min, before blocking in PBS/BSA 1%/Triton 0.2%.

  2. Immunolabeling of slices
    1. Continue using the same Eppendorf tubes containing slices and mount them on a turning wheel during incubations with primary and secondary antibodies and during washings.
    2. Incubation with first antibodies diluted in 1 ml PBS/BSA 1%/Triton 0.2% is overnight in a cold room at 4 °C.
    3. Rinse 3 x 10 min in 1 ml of PBS/BSA 1%/Triton 0.2%.
    4. Incubate with 1 ml of secondary antibodies diluted in PBS/BSA 1%/Triton 0.2% (2 μl stock antibody supplemented with 2 μl stock Phalloidin-Alexa 568 per tube) for 5 h at RT.
    5. Rinse 2 x 10 min in.
    6. Incubate in 2 μl of stock DAPI diluted in PBS/BSA 1%/Triton 0.2% for 20 min.
    7. Rinse 1x in PBS for 10 min.
    8. Remove the PBS and replace by 150 μl of PBS. Resuspend the slices and reverse the tube to deposit its contents on a microscope slide positioned under the binocular.
    9. Using the U-100 Insulin syringe and needle, collect the slices in the center of the slide.
    10. Using a yellow tip on a micropipette and slightly tilting the slide, remove the PBS to leave the fragments almost dry. The last trace of PBS is removed with the help of a filter paper.
    11. Using a cut-off yellow tip, add 30 μl Prolong Gold mounting medium to the slices and suspend them into it using the U-100 Insulin syringe and needle.
    12. Carefully lower an 18 x 18 mm glass coverslip in order to mount the slices.
      Note: About half of the slices will lie on their side.
    13. Leave at RT overnight to allow the mounting medium to polymerize and store in the dark at 4 °C.

  3. Fast confocal microscopy
    In order to be able to collect a sufficiently large number of image stacks during one session, we recommend using a fast confocal microscope and a 63x NA 1.4 oil immersion lens. For example, use a Leica SP5 confocal microscope scanning in bidirectional resonance mode (8,000 Hz), with 8x averaging/plane/per channel. Detect DAPI and Alexa 647 simultaneously and Alexa 488 and 568 (or 555) sequentially. Rotate the scanning head so that one field comprises two to three crypts oriented parallel to the scanning direction. An acquisition typically consists of 60 planes x 4 channels, with a step of 0.5 μm and a pixel size of 141 nm.
    Numerous images and 3D animations can be consulted in the paper freely downloadable here: http://jcb.rupress.org/articleusage?rid=198/3/331

Recipes

  1. 2x PHEM (500 ml)
    18.14 g
    Pipes
    6.5 g
    HEPES
    3.8 g
    EGTA
    0.99 g
    MgSO4
    Dissolve in 500 ml water and adjust to pH 7.0 with 10 mM KOH
  2. Fixative (3% PFA-PHEM 1x-Triton 0.2% -Taxol 15 μM) (10 ml)
    1,875 ml
    16% PFA
    5 ml
    2x PHEM
    100 μl
    20% Triton-X-100
    38 μl 
    4 mM Taxol (dissolved in DMSO)
    3 ml
    H2O
  3. 20 mM Sodium citrate buffer, pH 6.0
    Dissolve 5.88 g Tri-sodium citrate dehydrate in 1,000 ml distilled water
    Adjust pH to 6.0 with 1 N HCl
  4. Solutions of primary affinity-purified antibodies
    Primary affinity-purified antibodies are diluted from stock to 2 to 6 μg antibody/ml in PBS/BSA 1%/Triton 0.2%. Usually, this corresponds to a dilution of 1/500 to 1/2,000.
    Note: Each antibody must be tested for specificity and the optimal antibody concentration determined by a serial dilution to achieve between 1 to 6 μg/ml antibody. Ideally, specificity is tested on tissue after suppression of the antigen by knock out or knock down. If the localization is known, obtaining that can suffice.
  5. Solutions of secondary antibodies
    Secondary antibodies are used at 2 μl/ml of the stock solution in PBS/BSA 1%/Triton 0.2%, to which 2 μl Phalloidin-Alexa 568 stock solution per ml were added.

Acknowledgments

This protocol has been developed and reported in Bellis et al. (2013). Support was given to J. Bellis by the Ligue Contre le Cancer.

References

  1. Barker, N., van Oudenaarden, A. and Clevers, H. (2012). Identifying the stem cell of the intestinal crypt: strategies and pitfalls. Cell Stem Cell 11(4): 452-460.
  2. Bellis, J., Duluc, I., Romagnolo, B., Perret, C., Faux, M. C., Dujardin, D., Formstone, C., Lightowler, S., Ramsay, R. G., Freund, J. N. and De Mey, J. R. (2012). The tumor suppressor Apc controls planar cell polarities central to gut homeostasis. J Cell Biol 198(3): 331-341.
  3. Bjerknes, M., Khandanpour, C., Moroy, T., Fujiyama, T., Hoshino, M., Klisch, T. J., Ding, Q., Gan, L., Wang, J., Martin, M. G. and Cheng, H. (2012). Origin of the brush cell lineage in the mouse intestinal epithelium. Dev Biol 362(2): 194-218. 
  4. Caldwell, C. M., Green, R. A. and Kaplan, K. B. (2007). APC mutations lead to cytokinetic failures in vitro and tetraploid genotypes in Min mice. J Cell Biol 178(7): 1109-1120.
  5. De Mey, J. and Freund, J.-N. (2013). Understanding epithelial homeostasis in the intestine: An old battlefield of ideas, recent breakthroughs, and remaining controversies. Tissue Barriers 1(2): e24965. 
  6. Fleming, E. S., Zajac, M., Moschenross, D. M., Montrose, D. C., Rosenberg, D. W., Cowan, A. E. and Tirnauer, J. S. (2007). Planar spindle orientation and asymmetric cytokinesis in the mouse small intestine. J Histochem Cytochem 55(11): 1173-1180.
  7. Itzkovitz, S., Lyubimova, A., Blat, I. C., Maynard, M., van Es, J., Lees, J., Jacks, T., Clevers, H. and van Oudenaarden, A. (2012). Single-molecule transcript counting of stem-cell markers in the mouse intestine. Nat Cell Biol 14(1): 106-114.
  8. Potten, C. S., Booth, C. and Pritchard, D. M. (1997). The intestinal epithelial stem cell: the mucosal governor. Int J Exp Pathol 78(4): 219-243.
  9. Potten, C. S. (1998). Stem cells in gastrointestinal epithelium: numbers, characteristics and death. Philos Trans R Soc Lond B Biol Sci 353(1370): 821-830.
  10. Quyn, A. J., Appleton, P. L., Carey, F. A., Steele, R. J., Barker, N., Clevers, H., Ridgway, R. A., Sansom, O. J. and Nathke, I. S. (2010). Spindle orientation bias in gut epithelial stem cell compartments is lost in precancerous tissue. Cell Stem Cell 6(2): 175-181.
  11. Schliwa, M. and van Blerkom, J. (1981). Structural interaction of cytoskeletal components. J Cell Biol 90(1): 222-235.

材料和试剂

  1. 鼠标
  2. 异氟烷气体(Abbott Laboratories)
  3. 解剖盘蜡(黑色)(Fisher Scientific,目录号:S17432)
  4. 管道
  5. HEPES
  6. EGTA
  7. MgSO 4 4 /
  8. Triton-X-100
  9. 紫杉醇(来自Sigma-Aldrich的紫杉醇,目录号:T7191)
  10. PBS
  11. NaN 3
  12. 脱氧胆酸钠
  13. BSA 10%在PBS中
  14. 柠檬酸三钠二水合物(Merck KGaA,目录号:567446)
  15. 1N HCl(1mol/L)(Merck KGaA,目录号:1090571000)
  16. 多聚甲醛(PFA)16%溶液,EM级(Electron Microscopy Sciences,目录号:15710)
  17. 鬼笔环肽-Alexa 568(Life Technologies,Molecular Probes ,目录号:12380)
    注意:Alexa类型可以根据可用的其他荧光团来选择。
  18. DAPI:1mg/ml PBS中的储备溶液(在4℃下储存)
  19. Alexa标记的二抗。在我们的研究中,这些来自生命技术 - 分子探针山羊抗(物种)IgG(H + L)和所有高度交叉吸附,以确保不存在种间反应性。 注意:对于任何一次抗体的组合,必须选择相应的二级抗体和荧光团波长。在我们的例子中,Phalloidin-Alexa 568(红色荧光)用于标记由肌动蛋白制成的微丝,DAPI用于DNA,第二抗体通常用Alexa 488(绿色荧光)和Alexa 647(远红色荧光)标记。后者被推荐用于产生弱信号的抗原,因为其被目前的共聚焦显微镜系统非常好地检测。当不能使用鬼笔环肽时,可以另外使用Alexa 568或555(红色荧光)标记的二抗。
    注意:在我们的研究中,没有使用商业一抗。
  20. 不含DAPI的Prolong Gold封片剂(Life Technologies,目录号:P36934)
  21. 2x PHEM(参见配方)
  22. 固定剂(见配方)
  23. 20 mM柠檬酸钠缓冲液(见配方)
  24. 初级亲和纯化抗体的溶液(参见配方)
  25. 二抗溶液(参见配方)

设备

  1. 恒温水浴
  2. 用于异氟烷麻醉的Tem Sega蒸发器装置
  3. 剪刀3厘米和微型剪刀(精细科学工具)
  4. 镊子n°5(Fine Science Tools)
  5. 双目
  6. 30 G 1/2"针
  7. U-100胰岛素注射器+针
  8. 显微镜载片76×26×1.1mm
  9. 1.5 ml Eppendorf管
  10. 微量移液器
  11. 18 x 18 mm玻璃盖玻片 1
  12. 快速共聚焦显微镜(我们使用Leica SP5共聚焦显微镜)
  13. 63x NA 1.4油浸镜头

程序

  1. 样品制备
    1. 在组织收集期间需要麻醉小鼠。在麻醉前10分钟,在50ml塑料管中制备15ml PHEM 1x(来自PHEM 2x),并在恒温水浴中温热至37℃。将容器中的管在37℃的水中运输到解剖室,并在几分钟内使用
    2. 麻醉鼠标使用异氟醚吸入与Tem Sega蒸发器的帮助
    3. 在继续麻醉的同时,将其置于放置在化学萃取罩中的双目显微镜下,以便操作者不吸入异氟烷和甲醛气体。
    4. 用细剪刀打开腹部,相对于肛门定位远端(下降)结肠,将其从周围组织中取出,并与后者相距约2.5厘米。对于空肠,横切它从盲肠/近端结肠约2.5厘米。有关小鼠消化道的计划,请参阅: http://www.informatics。 jax.org/cookbook/figures/figure76.shtml。
      注意:对于我们的研究,敏感的基于细胞骨架的结构,如有丝分裂纺锤体的保存是必不可少的,只能通过快速处理实现。因此,我们每个肠段使用一个动物,并没有矫直肠。对于其他研究,例如某些细胞类型的核中存在某些转录因子,这可能不太重要。
    5. 使用注射器,用PHEM 1x在37℃冲洗结肠或空肠切片。
      注意:37℃对于避免对有温度敏感的有丝分裂纺锤体的改变是重要的。对于其他研究,这可能不那么重要。
    6. 立即用固定剂补充15μM紫杉醇。
      注意:固定剂含有PHEM缓冲液,已知可优化Ca 2 + 敏感微管的保存(Schliwa和van Blerkom,1981 )和一些Triton-X-100以加速固定的速度。它补充有紫杉醇,以进一步防止微管损失。紫杉醇确实与微管结合,并使其对甲醛固定的降解具有抗性。没有添加紫杉醇,发现锭子更短,有时变形,使得我们的研究中所描述的锭子角度的测量不可靠。当微管的保存不是必需的时,可以省略紫杉醇。然而,推荐使用PHEM缓冲液,因为它可以防止甲醛固定期间的pH变化,例如PBS。
    7. 切出一个1-2厘米的部分,并将其放在充满硬化的解剖盘蜡(黑色)充满5ml固定剂在室温下的杯子。用细剪刀沿其长度切开结肠或空肠段。
    8. 通过用剪刀切割通过隔膜进入心脏牺牲鼠标
    9. 针组织扁平和轻微拉伸蜡表面,粘膜上,并继续在室温固定40分钟。
    10. 在约10分钟后,在固定剂中,在微型剪刀的帮助下将组织切成小的1.5mm 3 方块。
    11. 将它们转移到1.5毫升的Eppendorf管,总共40分钟后,在PBS中冲洗他们三次,每次10分钟。它们现在可以在4℃下在补充有8mM NaN 3的PBS中储存数周。
    12. 在微量移液管上使用截止的蓝色尖端,将几片转移到放置在双目下的显微镜载玻片上。对于结肠碎片,使用固定在U-100胰岛素注射器上的两个薄的30G 1/2"针去除肌肉衬里。为此,肌肉衬里必须首先定位在粘膜层下面。然后通过在层之间插入一根针并使用它将肌肉层固定在适当位置,同时使用另一根针滑动或剥离粘膜,将它们分开。
      注意:空肠的肌肉薄层很薄,很脆弱,不需要去除。
    13. 使用其中一个针作为切割装置,同时将粘膜碎片保持在适当位置,另一个切割成薄的约1.5mm长的切片,其包含两至三排邻接的隐窝。转移30-40这样切片到1.5ml离心管中。所有后续步骤在这样的Eppendorf管中进行,每个一抗联合一个
    14. 将管具有约30个粘膜切片在旋转的轮上,并在30分钟内连续孵育1ml含有200mM NH 4 Cl,3%脱氧胆酸钠的H 2中的PBS 0.5%Triton-X-100的PBS和PBS/BSA 1%/Triton 0.2%封闭。在每个温育步骤后,使用安装在微量移液管上的1ml蓝色吸头以在切片由于重力沉降到管底部之后去除液体。注意避免因靠着光源握住管子而损失切片。
      注意:必须制备H 2 O中的脱氧胆酸盐溶液。
    15. 对于用某些抗体(例如针对转录因子Atoh1或Cdx2)的标记,通过将含有样品的管与1ml的0.1mM柠檬酸钠缓冲液(pH6.0)(从20x储备缓冲液制备)置于块加热器在95℃下30分钟,然后在PBS/BSA 1%/Triton 0.2%中封闭
  2. 切片的免疫标记
    1. 继续使用含有切片的相同的Eppendorf管,并在与第一抗体和第二抗体孵育期间以及在洗涤期间将它们安装在转向轮上。
    2. 与在1ml PBS/BSA 1%/Triton 0.2%中稀释的第一抗体的孵育在4℃的冷室中过夜。
    3. 在1ml PBS/BSA 1%/Triton 0.2%中冲洗3×10分钟
    4. 与在PBS/BSA 1%/Triton 0.2%中稀释的1ml第二抗体(每管补充2μl储备Phalloidin-Alexa 568的2μl储备抗体)孵育5小时。
    5. 冲洗2×10分钟。
    6. 在2μl稀释在PBS/BSA 1%/Triton 0.2%的储备DAPI中孵育20分钟
    7. 在PBS中冲洗1×10分钟。
    8. 取出PBS,并用150μlPBS替换。 重新悬浮切片,倒转管,将其内容物放置在双目下的显微镜载玻片上。
    9. 使用U-100胰岛素注射器和针头,收集玻片中心的切片。
    10. 使用微量移液管上的黄色尖端并稍微倾斜载玻片,移除PBS以使碎片几乎干燥。 用滤纸帮助除去PBS的最后痕迹。
    11. 使用截止的黄色提示,添加30微升Prolong Gold封固介质到切片,并使用U-100胰岛素注射器和针将它们悬浮在它。
    12. 小心地降低一个18 x 18毫米玻璃盖玻片,以安装切片。
      注意:大约一半的切片会在他们身边。
    13. 在室温下放置过夜,使固定介质在4℃的黑暗中聚合并储存。

  3. 快速共聚焦显微镜
    为了能够在一个会话期间收集足够大数量的图像堆栈,我们建议使用快速共焦显微镜和63x NA 1.4油浸镜头。例如,使用Leica SP5共焦显微镜以双向共振模式(8,000Hz)扫描,每个通道具有8x平均/平面/平面。同时检测DAPI和Alexa 647,依次检测Alexa 488和568(或555)。旋转扫描头,使得一个场包括平行于扫描方向定向的两个至三个隐窝。采集通常由60个平面×4个通道组成,具有0.5μm的步长和141nm的像素尺寸。
    许多图片和3D动画都可以在这里免费下载: http://

食谱

  1. 2x PHEM(500ml)
    18.14克
    管道
    6.5克
    HEPES
    3.8克
    EGTA
    0.99克
    MgSO 4 4 /
    溶于500ml水中,用10mM KOH调至pH7.0
  2. 固定剂(3%PFA-PHEM 1X-Triton 0.2%-Taxol15μM)(10ml)
    1,875 ml
    16%PFA
    5 ml
    2x PHEM
    100微升
    20%Triton-X-100
    38μl 
    4mM紫杉醇(溶于DMSO)
    3 ml
    H sub 2 O
  3. 20mM柠檬酸钠缓冲液,pH6.0 将5.88g柠檬酸三钠脱水物溶解在1,000ml蒸馏水中
    用1N HCl调节pH至6.0
  4. 主要亲和纯化抗体的溶液
    将一级亲和纯化的抗体从储备液稀释至在PBS/BSA 1%/Triton 0.2%中的2至6μg抗体/ml。 通常,这对应于1/500到1/2,000的稀释 注意:必须测试每种抗体的特异性,并通过连续稀释测定最佳抗体浓度,以获得1至6μg/ml抗体。 理想地,在通过敲除或敲低抑制抗原后对组织测试特异性。 如果本地化是已知的,获得就足够了。
  5. 二抗溶液
    第二抗体以2μl/ml的PBS/BSA 1%/Triton 0.2%的储备溶液使用,向其中加入每ml2μl鬼笔环肽-Alexa 568储备液。

致谢

该协议已经在Bellis等人(2013)中开发和报道。 由Ligue Contrele癌症支持J.Bellis。

参考文献

  1. Barker,N.,van Oudenaarden,A。和Clevers,H。(2012)。 识别肠道隐窝的干细胞:策略和陷阱细胞 干细胞 11(4):452-460
  2. Bellis,J.,Duluc,I.,Romagnolo,B.,Perret,C.,Faux,MC,Dujardin,D.,Formstone,C.,Lightowler,S.,Ramsay,RG,Freund,JN和De Mey, JR(2012)。 肿瘤抑制因子 控制平面细胞极性,直接影响肠内平衡。/a> J Cell Biol 198(3):331-341
  3. Bjerknes,M.,Khandanpour,C.,Moroy,T.,Fujiyama,T.,Hoshino,M.,Klisch,TJ,Ding,Q.,Gan,L.,Wang, H.(2012)。 小鼠肠上皮中刷细胞谱系的起源。 362(2):194-218。
  4. Caldwell,C.M.,Green,R.A。和Kaplan,K.B。(2007)。 APC突变导致Min小鼠体外和四倍体基因型的细胞动力学失败。 J Cell Biol 178(7):1109-1120。
  5. De Mey,J.and Freund,J.-N. (2013年)。小肠中的上皮内平衡:一个老的战场的思想,最近的突破和剩余的争议。组织障碍1(2):e24965(2013)。 "。 target ="_ blank">了解肠道上皮内平衡:一个古老的思想战场,最近的突破和剩余的争议。 组织障碍 1(2):e24965。
  6. Fleming,E.S.,Zajac,M.,Moschenross,D.M.,Montrose,D.C.,Rosenberg,D.W.,Cowan,A.E.and Tirnauer,J.S。(2007)。 小鼠小肠中的平面主轴定向和不对称胞质分裂。 Histochem Cytochem 55(11):1173-1180。
  7. Itzkovitz,S.,Lyubimova,A.,Blat,I.C.,Maynard,M.,van Es,J.,Lees,J.,Jacks,T.,Clevers,H.and van Oudenaarden, 小鼠肠中干细胞标记物的单分子转录计数。 Nat Cell Biol 14(1):106-114。
  8. Potten,C.S.,Booth,C。和Pritchard,D.M。(1997)。 肠上皮干细胞:粘膜调节物。Int J Exp Pathol 78(4):219-243。
  9. Potten,C.S。(1998)。 胃肠上皮中的干细胞:数量,特征和死亡。 Philos Trans R Soc Lond B Biol Sci 353(1370):821-830。
  10. Quyn,A.J.,Appleton,P.L.,Carey,F.A.,Steele,R.J.,Barker,N.,Clevers,H.,Ridgway,R.A.,Sansom,O.J.and Nathke,I.S.(2010)。 肠上皮干细胞区室中的主轴方向偏差在癌前组织中丧失。 Cell Stem Cell 6(2):175-181。
  11. Schliwa,M。和van Blerkom,J。(1981)。 细胞骨架成分的结构相互作用 J Cell Biol 90 (1):222-235
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How to cite this protocol: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Bellis, J., Duluc, I., Freund, J. and De Mey, J. R. (2013). Immunolabelling of Thin Slices of Mouse Descending Colon and Jejunum. Bio-protocol 3(20): e942. DOI: 10.21769/BioProtoc.942; Full Text
  2. Bellis, J., Duluc, I., Romagnolo, B., Perret, C., Faux, M. C., Dujardin, D., Formstone, C., Lightowler, S., Ramsay, R. G., Freund, J. N. and De Mey, J. R. (2012). The tumor suppressor Apc controls planar cell polarities central to gut homeostasis. J Cell Biol 198(3): 331-341.




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