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Immunofluorescent Staining of Mouse Intestinal Stem Cells
小鼠小肠干细胞的免疫荧光染色   

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Abstract

Immunofluorescent staining of organoids can be performed to visualize molecular markers of cell behavior. For example, cell proliferation marked by incorporation of nucleotide (EdU), or to observe markers of intestinal differentiation including paneth cells, goblet cells, or enterocytes (see Figure 1). In this protocol we detail a method to fix, permeabilize, stain and mount intestinal organoids for analysis by immunofluorescent confocal microscopy.


Figure 1. A schematic depicting a crypt-villus forming organoid, and visualization of Paneth cells by immunofluorescence staining. Left: Small intestinal organoids grow as crypt-villus structures that contain all of the multiple differentiated lineages of the intestine. Right: Immunofluorescent staining can be used to visualize individual cell types in the organoid. Here paneth cells are visualized by staining for lysozyme (“Lyso,” Green), which reveals Paneth cells located at crypt bases. F-Actin (Red) reveals crypt structure at the apical surface of the epithelium, and DAPI (Blue) reveals cell nuclei. Scale bar is 25 μm.

Keywords: Immunofluoresescence(immunofluoresescence), Staining(染色), Mouse(鼠标), Intestinal stem cells(肠道干细胞), IF(如果)

Materials and Reagents

  1. 8 well chamber slides (Thermo Fisher Scientific, Lab-TekTM, catalog number: 154532 )
  2. Cover Glass, Rectangular #1 (24 x 50 mm, 0.12-0.16 mm) (Corning, catalog number: 2975-245 )
  3. Primary antibodies
    1. Rabbit anti-KRT20 (1:200) (Cell Signaling Technology, catalog number: 13063 )
    2. Rabbit anti-Lysozyme (1:200) (Dako, catalog number: EC 3.2.1.17 )
    3. Rabbit anti-Muc2 (1:200)/ VHL Antibody (M-20) (Santa Cruz Biotechnology, catalog number: H-300, sc-1534 )
    4. Villin Antibody (C-19) (1:200) (Santa Cruz Biotechnology, catalog number: sc-7672 )
  4. Secondary antibodies:
    1. Goat Anti-rabbit 568 (1:500) (Thermo Fisher Scientific, Molecular Probes, catalog number: 11036 )
      Note: Currently, it is “Thermo Fisher Scientific, NovexTM, catalog number: 11036”.
    2. Donkey Anti-goat 594 (1:500) (Thermo Fisher Scientific, Molecular Probes, catalog number: 11058 )
      Note: Currently, it is “Thermo Fisher Scientific, NovexTM, catalog number: 11058”.
    3. Goat Anti-rat 488 (1:500) (Thermo Fisher Scientific, Molecular Probes, catalog number: a11006 )
      Note: Currently, it is “Thermo Fisher Scientific, NovexTM, catalog number: a11006”.
  5. Phosphate Buffered Saline (PBS) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10010023 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 10010023”.
  6. Paraformaldehyde (PFA) 16% Solution, EM Grade (Electron Microscopy Sciences, catalog number: 15710-S )
  7. Freshly prepared 4% PFA in 1x PME Buffer
  8. ProLong Gold Antifade Mountant (Thermo Fisher Scientific, Molecular Probes, catalog number: P10144 )
    Note: Currently, it is “Thermo Fisher Scientific, ProLong®, catalog number: P10144”.
  9. Clear Nail Polish (available at local drug store)
  10. Optional
    1. Click-iT EdU Alexa Fluor 647 for Cell Proliferation (Thermo Fisher Scientific, Invitrogen, catalog number: C10340 )
      Note: Currently, it is “Thermo Fisher Scientific, Molecular ProbesTM, catalog number: C10340”.
    2. Alexa Fluor 647 Phalloidin (Thermo Fisher Scientific, Molecular ProbeTM, catalog number: A22287 )
    3. BCIP/NBT Substrate Kit (Vector Laboratories, catalog number: SK-5400 )
    4. 1 μg/ml DAPI for nucleic acid staining (Sigma-Aldrich, catalog number: D9542 )
      Note: Materials and equipment to grow organoids prior to fixation (see Isolation, culture, and maintenance of mouse intestinal stem cells)
  11. Tris(hydroxymethyl)aminomethane (Sigma-Aldrich, catalog number: 252859 )
  12. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
  13. PIPES (Sigma-Aldrich, catalog number: P6757 )
  14. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  15. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884 )
  16. TritonTM X-100 (Sigma-Aldrich, catalog number: X100 )
  17. TWEEN® 20 (Sigma-Aldrich, catalog number: P2287 )
  18. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2058 )
  19. Tris buffered saline (TBS) (see Recipes)
  20. 10x PME buffer (see Recipes)
  21. IF buffer (see Recipes)
  22. Blocking solution (see Recipes)
  23. Permeabilization solution (see Recipes)

Equipment

  1. Benchtop Multi-Purpose Rotator (Thermo Fisher Scientific, model: 2309 )
  2. Leica Inverted Confocal SP8 equipped with a White Light Laser, a Leica HyD Detector and the Leica Application Suite software

Software

  1. Leica Application Suite

Procedure

  1. Plate 40 μl of the matrigel containing organoids into 1 well each of an 8-well chamber-slide. After letting harden in the incubator for 10-15 min, add 400 μl of media to the well and change at least every two days. If analyzing proliferation by EdU incorporation, add 10 μM EdU to the wells 6 h prior to fixation.
  2. When ready for fixation, remove the media and add 300 μl freshly prepared 4% PFA in PME buffer (room temperature) for 20 min. For this step, and throughout the entire protocol, to change the solution in the well, carefully place a pipette tip into the side of the well and draw up the solution. Then carefully add the new solution down the side of the well. This is performed the same way when changing media (O’Rourke et al., 2016).
  3. Remove the fixative, wash once with IF buffer. Add 300 μl of Permeabilization Solution to the well to permeabilize for 20 min.
  4. Remove the solution, wash once with IF Buffer and then add Blocking Solution for 30 min at room temperature.
  5. Alternative step: To visualize enterocytes by alkaline phosphatase staining, wash the fixed cells twice with TBS and then incubate with 300 μl of the BCIP/NBT Substrate Kit Solution for 15 min in the dark (follow directions that accompany the kit to make the BCIP/NBT solution). The chambers are then washed twice with TBS and then imaged using bright field microscopy. An example is shown in Figure 2.


    Figure 2. A brightfield image of a small intestinal organoid showing strong alkaline phosphatase activity (indicating the presence of differentiated enterocytes). Scale bar is 50 μm.

  6. Alternative step: Prior to primary antibody incubation, the samples can be processed for EdU visualization via Click-It chemistry using the directions provided with the kit. At this point follow the Invitrogen Protocol step 4.3 and finish with 4.7. Then, if required, incubate your sample in 150 μl of primary antibody as in step 7. For an example of an EdU and Keratin 20 stained small intestinal organoid, see Figure 3. Be sure to protect the sample from light from this step forward.
  7. Prepare the primary antibody in blocking solution and add 150 μl to each well. See Notes on Materials for antibodies we have validated. Let the chamber-slide sit overnight in a large, humidified chamber at 4 °C. Do not leave the chamber-slide lid on the chamber-slide as it can cause liquid to be pulled out of the chamber.
  8. After overnight incubation, wash each well 3 times for 5 min each with 300 μl IF buffer. Place the chamber slide on a benchtop orbital shaker set to 60 RPM during each wash.
  9. Prepare secondary antibody in Blocking Solution. For a list of secondary antibodies we have validated, see Note 4. Add 150 μl of the solution to the well and let sit at room temperature for 1 h, being sure to protect from light. Optionally, a phalloidin-conjugated fluorophore can be added to the secondary solution to visualize F-actin polymers.
  10. Optional step: To visualize DNA/nuclei, prepare a solution of DAPI (1 μg/ml) in IF buffer and add 300 μl to each well for 5 min. Then proceed with washing (step 11).
  11. After secondary incubation, wash each well 3 times with 300 μl IF buffer as in step 8.
  12. Remove the IF buffer and detach the chambers using the removal kit that accompanies the chamber slides.
  13. Using a p200 pipette tip with the tip cut off (see Note 6), add a small amount of ProLong Gold antifade mounting medium over each specimen (~20-30 μl each).
  14. Place a cover slip over the specimen, avoiding bubbles. Use clear nail polish to seal the sides of the cover glass to the slide. Let harden overnight at room temperature in the dark. After imaging, the slide can be stored at 4 °C for at least 6 months with minimal loss of fluorescence.


    Figure 3. Immunofluorescent image of a small intestinal organoid showing markers of proliferation (EdU, Red), differentiation (Krt20, Green) and nuclei (blue). Scale bar is 50 μm.

Notes

  1. During washes you will need to pipette liquid in and out of the chamber wells while carefully avoiding the organoids. A p1000 pipette tip with a tapered end can be helpful for this.
  2. We have had success with the Click-iT Edu Alexa Fluor 647 kit from Invitrogen, but note this kit is available with a wide range of fluorophores, which can be used to prevent overlap with any fluorescent protein that may be native to your cells. Note that this kit will also destroy F-actin, which prevents visualization of the actin cytoskeleton by Phalloidin conjugated fluorophores, as well as native GFP.
  3. We have validated the following primary antibodies for use in this assay: Rabbit anti-KRT20 (1:200), rabbit anti-Lysozyme (1:200), rabbit anti-Muc2 (1:200) and goat anti-Villin (1:200).
  4. We have validated the following secondary antibodies for use in this assay: Anti-rabbit 568 (1:500), anti-goat 594 (1:500) and anti-rat 488 (1:500).
  5. After the fixation and permeabilization steps, most of the Matrigel will have degraded, leaving the organoids to rest on the bottom of the well. This is true for small and large intestinal organoids grown for 4-6 days in culture prior to fixation, perhaps due to the production of intestinal proteases. We have noticed that shorter culture times, or different organoid cell types, can prevent this.
  6. When using the p200 to pipette the mounting medium over the cells, it is helpful to first use scissors to cut the tip at the tapered end to make it larger in diameter, which will avoid bubbles when pipetting the solution.
  7. We have had the best results imaging these samples using a confocal microscope. Specifically, we use a Leica Inverted Confocal SP8 equipped with a White Light Laser, a Leica HyD Detector and the Leica Application Suite software. This setup allows the excitation laser to be set to any wavelength between 470 and 670 nm, in addition to a 405 laser for DAPI excitation and the standard argon laser lines of 458, 476, 488, 496 and 514 nm. This means one can optimally excite and cleanly separate any fluorophores. The SP8 is also equipped with gating technology, which significantly eliminates autofluorescence.
  8. We have used the HC PL APO 20x .7NA IMM and the HCX PL APO 40x 1.1NA objectives for imaging these samples.
  9. For an example of an immunofluorescent image obtained using this microscope setup (see Figure 3).

Recipes

  1. Tris buffered saline (TBS)
    25 mM Tris
    0.15M NaCl
    Final pH of solution should be 7.2 to 7.5
  2. 10x PME buffer
    500 mM PIPES
    25 mM MgCl2
    50 mM EDTA
  3. IF buffer
    PBS containing:
    1. 0.2% Triton X-10
    2. 0.05% Tween
  4. Blocking solution
    IF Buffer containing:
    1% bovine serum albumin (BSA)
  5. Permeabilization solution
    PBS containing
    0.5% Triton X-100

Acknowledgments

We thank members of the Lowe Lab, as well as the Molecular Cytology Core Facility at Memorial Sloan Kettering Cancer Center, for helpful input. This work was supported by a program project grant from the NIH/ NCI (CA-013106). L. E. D. was supported by a National Health and Medical Research Council (NHMRC) Overseas Biomedical Fellowship and a K22 Career Development Award from the NCI/NIH (CA-181280-01). K. P. O. was supported by a Medical Scientist Training Program grant from the National Institute of General Medical Sciences of the NIH under award number T32GM07739 to the Weill Cornell/Rockefeller/Sloan-Kettering Tri-Institutional MD-PhD Program. S. W. L. is the Geoffrey Beene Chair of Cancer Biology and an investigator of the Howard Hughes Medical Institute.

References

  1. Dow, L. E., O'Rourke, K. P., Simon, J., Tschaharganeh, D. F., van Es, J. H., Clevers, H. and Lowe, S. W. (2015). Apc restoration promotes cellular differentiation and reestablishes crypt homeostasis in colorectal cancer. Cell 161(7): 1539-1552.
  2. O’Rourke, K. P., Ackerman, S., Dow, L. E. and Lowe, S. W. (2016). Isolation, culture, and maintenance of mouse intestinal stem cells. Bio-protocol 6(4): e1733.

简介

可以进行类器官的免疫荧光染色以显现细胞行为的分子标志物。例如,通过掺入核苷酸(EdU)标记的细胞增殖,或观察肠分化的标志物,包括paneth细胞,杯状细胞或肠细胞(参见图1)。在这个协议中,我们详细的方法来修复,透化,染色和安装肠组织,通过免疫荧光共聚焦显微镜分析。


图1.描绘隐窝 - 绒毛形成类器官的示意图,通过免疫荧光染色观察Paneth细胞。肠器官类生长为含有所有肠的多种分化谱系的隐窝 - 绒毛结构。右:免疫荧光染色可用于显现器官类型中的单个细胞类型。通过染色溶菌酶("Lyso,"Green)显示paneth细胞,其显示位于隐窝碱基的Paneth细胞。 F-肌动蛋白(红色)显示在上皮的顶端表面的隐窝结构,DAPI(蓝色)揭示细胞核。比例尺为25μm。

关键字:immunofluoresescence, 染色, 鼠标, 肠道干细胞, 如果

材料和试剂

  1. 8孔腔室玻片(Thermo Fisher Scientific,Lab-Tek TM,目录号:154532)
  2. 盖玻片,矩形#1(24×50mm,0.12-0.16mm)(Corning,目录号:2975-245)
  3. 一抗
    1. 兔抗KRT20(1:200)(Cell Signaling Technology,目录号:13063)
    2. 兔抗溶菌酶(1:200)(Dako,目录号:EC 3.2.1.17)
    3. 兔抗Muc2(1:200)/VHL抗体(M-20)(Santa Cruz Biotechnology,目录号:H-300,sc-1534)
    4. Villin抗体(C-19)(1:200)(Santa Cruz Biotechnology,目录号:sc-7672)
  4. 次级抗体:
    1. 山羊抗兔568(1:500)(Thermo Fisher Scientific,Molecular Probes,目录号:11036)
      />
    2. 驴抗山羊594(1:500)(Thermo Fisher Scientific,Molecular Probes,目录号:11058)
      />
    3. 山羊抗大鼠488(1:500)(Thermo Fisher Scientific,Molecular Probes,目录号:a11006)
      注意:目前,"Thermo Fisher Scientific,Novex TM ,目录号:a11006"
  5. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Invitrogen TM ,目录号:10010023)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:10010023" />
  6. 多聚甲醛(PFA)16%溶液,EM级(Electron Microscopy Sciences,目录号:15710-S)
  7. 新鲜制备的4%PFA在1x PME缓冲液中
  8. ProLong Gold Antifade Mountant(Thermo Fisher Scientific,Molecular Probes,目录号:P10144)
    注意:目前,是"Thermo Fisher Scientific,ProLong ,目录号:P10144" />
  9. 清澈指甲油(可在当地药店买到)
  10. 可选
    1. 用于细胞增殖的Click-iT EdU Alexa Fluor 647(Thermo Fisher Scientific,Invitrogen,目录号:C10340)
      注意:目前,"Thermo Fisher Scientific,Molecular Probes TM ,目录号:C10340" br />
    2. Alexa Fluor 647鬼笔环肽(Thermo Fisher Scientific,Molecular Probe ,目录号:A22287)
    3. BCIP/NBT底物试剂盒(Vector Laboratories,目录号:SK-5400)
    4. 1μg/ml用于核酸染色的DAPI(Sigma-Aldrich,目录号:D9542) 注意:在固定之前生长器官类的材料和设备(见 小鼠肠干细胞的分离,培养和维持)
  11. 三(羟甲基)氨基甲烷(Sigma-Aldrich,目录号:252859)
  12. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9888)
  13. PIPES(Sigma-Aldrich,目录号:P6757)
  14. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  15. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E9884)
  16. TritonX-100(Sigma-Aldrich,目录号:X100)
  17. TWEEN 20(Sigma-Aldrich,目录号:P2287)
  18. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2058)
  19. Tris缓冲盐水(TBS)(参见Recipes)
  20. 10x PME缓冲区(请参阅配方)
  21. IF缓冲区(参见配方)
  22. 阻止解决方案(参见配方)
  23. 渗透溶液(参见配方)

设备

  1. 台式多用途旋转器(Thermo Fisher Scientific,型号:2309)
  2. 配备白光激光器,Leica HyD检测器和Leica Application Suite软件的Leica Inverted Confocal SP8系统

软件

  1. Leica应用程序套件

程序

  1. 将40μl含有类器物的基质胶加入1孔,每孔8孔室 - 载玻片。让孵化器硬化10-15分钟后,向孔中加入400μl培养基,至少每两天更换一次。如果通过EdU掺入来分析增殖,在固定前6小时向孔中加入10μMEdU
  2. 当准备固定,删除介质,加入300μl新鲜制备的4%PFA在PME缓冲液(室温)20分钟。对于这一步,并在整个协议,以更改在井中的解决方案,小心地将移液器吸头放入井的一侧,并提出解决方案。然后小心地添加新的解决方案在井的侧面。这在改变媒体时以相同的方式执行(O'Rourke等人,2016)。
  3. 取出固定液,用IF缓冲液洗一次。加入300微升渗透溶液到孔中透化20分钟
  4. 取出溶液,用IF Buffer洗涤一次,然后在室温下加入封闭溶液30分钟
  5. 备选步骤:通过碱性磷酸酶染色可视化肠细胞,用TBS洗涤固定的细胞两次,然后与300μl的BCIP/NBT底物试剂盒溶液在黑暗中孵育15分钟(按照试剂盒附带的指示,使BCIP/NBT溶液)。然后将室用TBS洗涤两次,然后使用明视野显微镜成像。一个例子如图2所示。


    图2.显示强碱性磷酸酶活性(表明存在分化的肠细胞)的小肠组织样的明场图像。比例尺为50μm。

  6. 备选步骤:在一抗孵育前,可以使用试剂盒提供的指导,通过Click-It化学处理样品以进行EdU可视化。此时,请遵循 Invitrogen Protocol 步骤4.3,并以4.7结束。然后,如果需要,孵育你的样品在150微升的一抗在步骤7.对于一个EdU和角蛋白20染色的小肠类器官的例子,见图3.确保保护样品免受这一步的光。
  7. 准备一抗在封闭溶液,并添加150微升到每个孔。请参阅有关我们已验证的抗体的材料说明。让室 - 幻灯片在4℃下在大的湿润室中静置过夜。不要将腔室滑动盖留在腔室滑板上,因为它可能导致液体从腔室中拉出
  8. 孵育过夜后,用300μlIF缓冲液洗涤每个孔3次,每次5分钟。将室滑板放置在台式轨道摇床上,在每次洗涤期间设??定为60RPM。
  9. 在封闭液中制备二抗。对于我们已经验证的二次抗体的列表,参见注释4.将150μl溶液加入孔中,并在室温下放置1小时,以确保避光。任选地,可将鬼笔环肽缀合的荧光团加入到第二溶液中以显现F-肌动蛋白聚合物。
  10. 可选步骤:为了可视化DNA /核,制备DAPI(1μg/ml)在IF缓冲液中的溶液,并向每个孔中加入300μl5分钟。然后继续洗涤(步骤11)。
  11. 次级培养后,用步骤8中的300μlIF缓冲液洗涤每个孔3次
  12. 取出IF缓冲液,并使用拆卸试剂盒,随着腔室幻灯片分离腔室。
  13. 使用尖端切断的p200移液器吸头(见注6),在每个样品上加入少量ProLong Gold抗衰老封固剂(每个约20-30μl)。
  14. 将盖玻片放在样品上,避免气泡。使用清晰的指甲油密封玻璃盖玻片的两侧。让在室温下在黑暗中硬化过夜。成像后,载玻片可以在4℃下储存至少6个月,同时荧光损失最小

    图3.显示增殖(EdU,红色),分化(Krt20,绿色)和细胞核(蓝色)的标志物的小肠组织样的免疫荧光图像。比例尺为50μm。

笔记

  1. 在洗涤过程中,您需要将液体吸入和排出腔室,同时小心避免类器官。具有锥形端的p1000移液管尖端可以有助于此
  2. 我们已经从Invitrogen的Click-iT Edu Alexa Fluor 647试剂盒获得了成功,但是请注意,该试剂盒提供多种荧光团,可用于防止与任何可能对您的细胞天然的荧光蛋白重叠。请注意,这个工具包还将销毁F-肌动蛋白,防止肌动蛋白细胞骨架的Phalloidin共轭荧光团,以及原生GFP的可视化。
  3. 我们验证了用于该测定的以下初级抗体:兔抗KRT20(1:200),兔抗溶菌酶(1:200),兔抗Muc2(1:200)和山羊抗绒毛蛋白(1: 200)。
  4. 我们已经验证了用于该测定的以下二抗:抗兔568(1:500),抗山羊594(1:500)和抗大鼠488(1:500)。
  5. 在固定和透化步骤之后,大部分Matrigel将会降解,使得类器物停留在孔的底部。这对于在固定之前在培养物中生长4-6天的小和大肠组织样品是真的,这可能是由于肠蛋白酶的产生。我们注意到,较短的培养时间或不同的器官类型细胞类型可以预防这种情况
  6. 当使用p200将吸附介质吸移到细胞上时,首先使用剪刀在锥形末端切割尖端以使其直径更大,这有助于移除溶液时避免气泡。
  7. 我们已经有使用共焦显微镜成像这些样品的最佳结果。具体来说,我们使用配备白光激光器,Leica HyD检测器和Leica Application Suite软件的Leica Inverted Confocal SP8。除了用于DAPI激发的405激光器和458,476,488,496和514nm的标准氩激光线之外,该设置允许将激发激光设置在470和670nm之间的任何波长。这意味着可以最佳地激发和干净地分离任何荧光团。 SP8还配备了门控技术,可显着消除自身荧光。
  8. 我们使用HC PL APO 20x .7NA IMM和HCX PL APO 40x 1.1NA物镜对这些样品进行成像。
  9. 关于使用该显微镜装置获得的免疫荧光图像的实例(参见图3)

食谱

  1. Tris缓冲盐水(TBS)
    25 mM Tris
    0.15M NaCl
    溶液的最终pH应为7.2至7.5
  2. 10x PME缓冲区
    500 mM PIPES
    25mM MgCl 2·h/v 50 mM EDTA
  3. IF缓冲区
    包含
    的PBS
    1. 0.2%Triton X-10
    2. 0.05%吐温
  4. 封锁解决方案
    IF缓冲区包含:
    1%牛血清白蛋白(BSA)
  5. 渗透溶液
    含有
    的PBS 0.5%Triton X-100

致谢

我们感谢Lowe实验室的成员,以及Memorial Sloan Kettering癌症中心的分子细胞学核心设施,为您提供帮助。这项工作得到了来自NIH/NCI(CA-013106)的计划项目赠款的支持。 L.E.D。获得国家卫生和医学研究委员会(NHMRC)海外生物医学奖学金和来自NCI/NIH(CA-181280-01)的K22职业发展奖。 K.P. O.得到NIH的国家综合医学科学研究所授予的医学科学家培训计划资助,授予T32GM07739授予Weill Cornell /洛克菲勒/Sloan-Kettering三机构MD-PhD计划。 S. W. L.是Geoffrey Beene癌症生物学主席和霍华德休斯医学研究所的研究员。

参考文献

  1. Dow,L.E.,O'Rourke,K.P.,Simon,J.,Tschaharganeh,D.F.,van Es,J.H.,Clevers,H.and Lowe,S.W。(2015)。 Apc恢复促进细胞分化,恢复结肠直肠癌中的隐窝动态平衡。细胞 161(7):1539-1552。
  2. O'Rourke,K.P.,Ackerman,S.,Dow,L.E。和Lowe,S.W。(2016)。 小鼠肠干细胞的分离,培养和维持 生物协议 6(4):e1733。
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引用:O’Rourke, K. P., Dow, L. E. and Lowe, S. W. (2016). Immunofluorescent Staining of Mouse Intestinal Stem Cells. Bio-protocol 6(4): e1732. DOI: 10.21769/BioProtoc.1732.
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