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Aug 2021

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TUNEL Labeling to Detect Double-stranded DNA Breaks in Caenorhabditis elegans Gonads
TUNEL 标记检测秀丽隐杆线虫性腺中的双链 DNA 断裂   

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Abstract

Analysis of DNA double strand breaks (DSBs) is important for understanding dyshomeostasis within the nucleus, impaired DNA repair mechanisms, and cell death. In the C. elegans germline, DSBs are important indicators of all three above-mentioned conditions. Although multiple methods exist to assess apoptosis in the germline of C. elegans, direct assessment of DSBs without the need for a reporter allele or protein-specific antibody is useful. As such, unbiased immunofluorescent approaches can be favorable. This protocol details a method for using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) to assess DNA DSBs in dissected C. elegans germlines. Germlines are co-labeled with DAPI to allow for easy assessment of DNA DSBs. This approach allows for qualitative or quantitative measures of DNA DSBs.


Graphic abstract:



Schematic for TUNEL labeling of C. elegans germlines.


Keywords: C. elegans (秀丽隐杆线虫), Germline (种系), TUNEL (TUNEL), Double strand breaks (双链断裂)

Background

Many stresses, nuclear or otherwise, can result in germline phenotypes in C. elegans, including reduced brood size or complete sterility. When trying to understand the mechanism resulting in germline defects, it is necessary to investigate the possibility of increased DNA double strand breaks that are likely to lead to apoptosis. Although techniques are well established to assess germline apoptosis [e.g., analysis of cell corpses; CED-3::GFP transgenes (Chen et al., 2016)] and DNA damage [e.g., LacZ reporters transgenes (Pontier and Tijsterman, 2009); immunolabeling for DSB repair markers, such as RAD-51 and RPA-1 (Bae et al., 2020; Hinman et al., 2021)] in the C. elegans germline, inclusion of a genetically-encoded reporter can be complicated or impossible in the case of genetic linkage. Therefore, biochemical approaches may offer more flexibility in assessment of DNA DSBs. As presented here, TUNEL labeling can identify DNA DSBs in nearly all C. elegans genetic backgrounds (excluding those without a gonad). This approach not only eliminates the need to generate new mutant strains containing the reporter, but allows for parallel preparation and analysis of multiple samples. This protocol is an adaptation of a protocol used in Parusel (2006) and exactly as described inKropp et al. (2021).

Materials and Reagents

  1. 25 × 75 mm Superfrost Plus Slides (Diagger, catalog number: EF15978Z), store at room temperature

  2. 25 × 25-1 Microscope Cover Glass (Fisherbrand, catalog number: 12-542-C), store at room temperature

  3. 23 G 1¼ Precision Glide Needle (BD Bioscience, catalog number: 305120), store at room temperature

  4. KimWipes (Kimtech Science, Kimberly-Clark Professional, catalog number: 05511), store at room temperature

  5. M9 Buffer (IPM Scientific, catalog number: 11006-517), store at room temperature

  6. 10× PBS (KD Medical, catalog number: RGF-3210), store at room temperature

  7. Poly-L-lysine solution (Sigma Life Science, catalog number: P8920-100mL), store at room temperature

  8. Paraformaldehyde Powder (Sigma-Aldrich, catalog number: 158127), store at 4°C

  9. Triton X-100 (Sigma Life Science, catalog number: X100-100mL), store at room temperature

  10. Sodium Citrate tribasic dihydrate powder (Sigma-Aldrich, catalog number: C8532), store at room temperature

  11. TUNEL Assay Kit – FITC (Abcam, catalog number: ab66108), store at 4°C and -20°C

  12. DAPI powder (Sigma-Aldrich, catalog number: D9542), store at 4°C

  13. Vectashield (Vector Laboratories, Inc, catalog number: H-1000), store at 4°C

  14. Immersion oil for microscopy (Cargille, catalog number: 16482), store at room temperature


Stock solutions to be made
  1. 4% paraformaldehyde (PFA) solution (see Recipes).

  2. PTX solution (see Recipes)

  3. 100 mM sodium citrate buffer (see Recipes)

  4. DAPI solution (see Recipes)

  5. TUNEL reaction mix from TUNEL Assay kit (see Recipes)

  6. 1 M Sodium citrate (see Recipes)

Equipment

  1. SMZ645 stereomicroscope (Nikon, model: SMZ645-L)

  2. Digital Heat block (VWR Scientific Products, catalog number: 13259-050)

  3. Spinning disk confocal microscope

    1. Nikon Eclipse Ti2-E inverted microscope

    2. Yokagawa CSUZ-1 spinning disk

    3. Photometrics Prime95B camera

    4. Nikon Plan Flour 40×/1.3 oil immersion objective

Software

  1. Elements Advanced Research (Nikon, https://www.microscope.healthcare.nikon.com/products/software/nis-elements/nis-elements-advanced-research)

Procedure

  1. Working reagent preparation

    1. Prepare Poly-L-lysine coated slides

      1. Add 3–5 μL of Poly-L-lysine to the center of a Superfrost Plus Slide.

      2. Spread the drop of Poly-L-lysine solution into an even layer ~1 cm in diameter with the side of a pipet tip.

      3. Dry at room temperature (RT) overnight. Slides can be stored at 4°C for up to one month once dry.

      4. Allow slides to come to RT prior to the procedure.

    2. Prepare 4% paraformaldehyde (PFA) solution (see Recipes).

    3. Prepare PTX solution (see Recipes).

    4. Prepare 100 mM sodium citrate buffer (see Recipes).

    5. Prepare DAPI solution (see Recipes). Excess solution can be stored at 4°C for up to one month, protected from light.



    Figure 1. Picking animals from plate to slide.

    (A) Using a platinum wire pick, pick the animal(s) from the culture plate to a watch glass containing M9 buffer. (B) Using a sweeping motion, gently remove the animal(s) from the pick into the M9 buffer in the watch glass. (C) Using a sweeping motion, gently lift individual animals from the watch glass and transfer them to a slide containing a drop of M9 buffer. (D) Representative schematic of a slide with an individual animal contained within a droplet of M9 buffer on a dried bed of poly-L-lysine. Representations are not to scale.


  2. Sample preparation

    1. From a synchronous population of C. elegans (L4 or adult), manually pick individual animals into a watch glass containing M9 solution, to remove excess bacteria (Figure 1A and 1B).

    2. Pipet 10 μL of M9 solution to the Poly-L-lysine-coated region of a slide.

    3. Individually pick C. elegans from the watch glass into the droplet of M9 on the slide (Figure 1C and 1D). Add ~10 animals to the droplet in total.

      Note: When picking worms from the watch glass, gently scoop them up individually with your pick. The surface tension of the M9 will allow you to pick up individual worms. Remove them from the pick by gently sweeping the pick through the droplet of M9 on the slide.

    4. On the slide, dissect the animals’ gonads

      1. Using the edge of a needle, cut the head and/or tail from C. elegans on the slide. Gonads will automatically extrude and stick to the Poly-L-lysine coating (Figure 2).

        Notes:

        1) Some people prefer to use two needles and a scissoring motion, although we found that to be unnecessary.

        2) Addition of an anesthetic such as 1mM levamisole can be helpful.

        3) A video protocol can be found at https://www.jove.com/v/20131/c-elegans-gonad-extrusion-a-rapid-dissection-technique (Gervaise and Arur, 2016).

      2. Work quickly, as all gonads should be dissected within two minutes.



      Figure 2. Gonad dissection from adult C. elegans.

      (A) Representative image through a dissecting microscope of a day-one adult C. elegans with a 23 G 1¼ needle for scale. The tip of the needle is adjacent to the junction of the pharynx and intestine (also indicated by change from light to dark color). This is where the incision should be made. (B) Representative image of an extruded gonad as viewed through a dissecting microscope. The intestine will also extrude. Note that the head was not completely removed in this instance. (C) Enhanced image (from B) of extruded day-one adult gonad with regions labeled. MZ: Mitotic zone; TZ: Transition zone. Scale bars (A and B): 500 µm; (C) 100 µm.


    5. Fix sample with PFA

      1. Wick away excess M9 solution with the edge of a KimWipe (Figure 3).

      2. Pipet 25 μL of 4% PFA onto the sample. Gently add cover glass to prevent evaporation.

      3. Incubate at RT for 20 min.

      4. Carefully remove cover glass. Carefully remove PFA with a pipet and dispose in an appropriate receptacle.

        Notes:

        1) It may be necessary to use a razor blade to create a separation between the cover glass and the slide. If used, be sure not to damage the sample.

        2) PFA is a hazardous waste material. Dispose of it in accordance with your institution’s guidelines. All PTX washes immediately after PFA fixation should be assumed to contain PFA and disposed of with the PFA.

      5. Wash the sample with 25–50 μL of PTX solution three times for three min each. Use the same pipetting technique as used for PFA, to avoid damaging the samples. Dispose of PTX washes in the same receptacle as PFA.



      Figure 3. Schematic for fixing, washing, and staining extruded gonads.

      The animals will be contained within a droplet of buffer. Pipet or wick away (with a KimWipe) buffers at the end of each step. Add buffers with a pipet at the beginning of each step. To avoid disrupting the samples, pipet or wick from the edge of the buffer droplet as indicated by the area in the red oval. If necessary, tilt the slide to make wicking easier. Representations not to scale.


    6. Incubate sample in prepared sodium citrate buffer to improve antigen retrieval

      1. Pipet 25 μL of 10 mM sodium citrate solution on the sample and cover with cover glass.

      2. Place the slide on a heat block prewarmed to 65°C. Incubate for 20 min.

      3. Carefully remove cover glass. Wick away sodium citrate solution with the edge of KimWipe.

      4. Wash three times with PTX, as in step 5e. The PTX wash solution does not need special disposal at this point.

    7. Incubate sample in TUNEL reaction mix

      1. Prepare TUNEL reaction mix from TUNEL Assay kit (see Recipes).

      2. Pipet 51 μL of reaction mix onto the sample. Add cover glass to prevent evaporation.

      3. Incubate in a humid chamber at 37°C for 60 min. Protect the slide from light from this point forward.

        Note: Any plastic container with a damp paper towel is sufficient to make a humid chamber. If there is no natural way to keep the slide from sitting directly on the paper towel, inclusion of something to prop up the slide (such as two parallel pieces of a serological pipet) works well.

      4. Wash three times with PTX as in step 5e. The PTX wash solution does not need special disposal at this point.

    8. DAPI co-labeling

      1. Wick away PTX solution with the edge of a KimWipe.

      2. Add 25 μL of DAPI solution to sample and incubate at RT protected from light for 5 min

      3. Wash three times with PTX as in step 5e. The PTX wash solution does not need special disposal at this point.

    9. Mounting and cover slipping

      1. Wick away any remaining PTX solution with the edge of a KimWipe.

      2. Add one drop of Vectashield directly to the sample.

      3. Gently lower a clean cover glass onto the sample. To avoid bubbles, lower one edge first, then slowly lower the rest of the cover glass.

        Note: If bubbles are present, gently press on the bubble with a toothpick to move it to the edge of the cover glass.

      4. Wick away any excess Vectashield with a KimWipe. Seal cover glass with nail polish.

      5. Sample can be imaged immediately or the following day. Imaging the following day tends to yield better results. If waiting to image, store the slide at 4°C.



    Figure 4. Representative maximum intensity projection of a wild-type L4 C. elegans gonad co-labeled with DAPI and TUNEL.

    DAPI-stained nuclei are labeled in blue and TUNEL-positive nuclei are labeled in green. As this is an L4 gonad, no oocytes are present. The different regions of the gonad are discernable based on nuclear (DAPI) structure. Due to crossover formation during meiosis (diplotene), nuclei will label with TUNEL. Artifactual labeling of mitotic nuclei with TUNEL has been reported in other systems, including human tissue sections (Labat-Moleur et al., 1997) and Drosophila ovaries (Qi and Calvi, 2016). DNA nicks during replication are likely the cause (Mets and Meyer, 2009). Apparent pale labeling in the middle pachytene region is an artifact from stitching two z-stacks into a single image. Scale bar: 100 μm.


  3. Confocal Imaging

    1. Preparation

      1. Ensure that all appropriate lasers (405 nm and 488 nm) and light sources are turned on and warm. Set the 405 nm laser to 35% power and 100 ms exposure. Set the 488 nm laser to 25.5% power and 100 ms exposure.

      2. Mount the slide onto the microscope stage.

      3. If using an oil immersion objective, add a small drop of oil onto the cover glass.

      4. Find the desired sample.

    2. Image acquisition

      1. Set z-slice thickness to 0.3 μm.

      2. Define z-stack to encompass the entire germline diameter.

      3. Capture the z-stack.

      4. If necessary, capture second z-stack to image entire germline.

Data analysis

Data analysis should be performed at the discretion of the experimenter depending on ultimate end use. We qualitatively assessed TUNEL labeling, but quantitative measurements could be generated by determining the percentage of TUNEL-positive nuclei as a function of total nuclei ((# TUNEL+)/(# DAPI+)) or the percentage of TUNEL-positive nuclei within a specific region of the gonad (e.g., pachytene). Comparison of the percentage of TUNEL-positive nuclei between wild-type and mutant samples would be appropriate.

Notes

A positive control to increase incidence of DSBs is exposure of C. elegans to UV light. We achieved this by placing plates with C. elegans on a standard UV light box for 20 min. Plates were seeded with OP50 bacteria, although we do not believe this is explicitly necessary.

Recipes

All buffers were purchased. Preparations of incubation and wash buffers are indicated in below.

  1. 4% paraformaldehyde (PFA) solution

    In H2O, from 16% stock. Use fresh. May require heating. Store at 4°C.

  2. PTX solution

    10× PBS stock and Triton X-100. 1× PBS + 0.4% Triton X-100. Use fresh.

  3. 100 mM sodium citrate buffer

    1 M stock + 0.1% Triton X-100. Use fresh.

  4. DAPI solution

    10 μg/mL in H2O.

  5. TUNEL reaction mix from TUNEL Assay kit

    10 μL of Reaction Buffer, 0.75 μL of TdT Enzyme, 8 μL of FITC-dUTP, and 32.25 μL of ddH2O.

  6. 1 M Sodium citrate

    In H2O. May require heating. Store at room temperature.

Acknowledgments

This work was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases (A.G.). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. This protocol is an adaptation of a protocol used in Parusel (2006) and exactly as described in Kropp et al. (2021).

Competing interests

The authors declare no competing interests.

References

  1. Bae, W., Park, J. H., Lee, M. H., Park, H. W. and Koo, H. S. (2020). Hypersensitivity to DNA double-strand breaks associated with PARG deficiency is suppressed by exo-1 and polq-1 mutations in Caenorhabditis elegans. FEBS J 287(6): 1101-1115.
  2. Chen, X., Wang, Y., Chen, Y. Z., Harry, B. L., Nakagawa, A., Lee, E. S., Guo, H. and Xue, D. (2016). Regulation of CED-3 caspase localization and activation by C. elegans nuclear-membrane protein NPP-14. Nat Struct Mol Biol 23(11): 958-964.
  3. Gervaise, A. L. and Arur, S. (2016). Spatial and Temporal Analysis of Active ERK in the C. elegans Germline. J Vis Exp(117): 54901.
  4. Hinman, A. W., Yeh, H. Y., Roelens, B., Yamaya, K., Woglar, A., Bourbon, H. G., Chi, P. and Villeneuve, A. M. (2021). Caenorhabditis elegans DSB-3 reveals conservation and divergence among protein complexes promoting meiotic double-strand breaks.Proc Natl Acad Sci U S A 118(33): e2109306118.
  5. Kropp, P. A., Wu, J., Reidy, M., Shrestha, S., Rhodehouse, K., Rogers, P., Sack, M. N. and Golden, A. (2021). Allele-specific mitochondrial stress induced by Multiple Mitochondrial Dysfunctions Syndrome 1 pathogenic mutations modeled in Caenorhabditis elegans. PLoS Genet 17(8): e1009771.
  6. Labat-Moleur, F., Guillermet, C., Lorimier, P., Robert, C., Lantuejoul, S., Brambilla, E. and Negoescu, A. (1998). TUNEL apoptotic cell detection in tissue sections: critical evaluation and improvement. J Histochem Cytochem 46(3): 327-334.
  7. Mets, D. G. and Meyer, B. J. (2009). Condensins regulate meiotic DNA break distribution, thus crossover frequency, by controlling chromosome structure. Cell 139(1): 73-86.
  8. Parusel, C. T. (2006). URI-1 is required for DNA stability in C. elegans. Development 133(4): 621-629.
  9. Pontier, D. B. and Tijsterman, M. (2009). A robust network of double-strand break repair pathways governs genome integrity during C. elegans development. Curr Biol 19(16): 1384-1388.
  10. Qi, S. and Calvi, B. R. (2016). Different cell cycle modifications repress apoptosis at different steps independent of developmental signaling in Drosophila. Mol Biol Cell 27(12): 1885-1897.


简介

[摘要] DNA 双链断裂 (DSB) 的分析对于了解细胞核内的动态平衡失调、DNA 修复机制受损和细胞死亡具有重要意义。在C. elegans种系中,DSB 是上述所有三种情况的重要指标。尽管存在多种方法来评估秀丽隐杆线虫种系中的细胞凋亡, 无需报告等位基因或蛋白质特异性抗体直接评估 DSB 是有用的。因此,无偏见的免疫荧光方法可能是有利的。该协议详细介绍了使用末端脱氧核苷酸转移酶d U TP的方法 尼克和标记( TUNEL ) 以评估解剖的秀丽隐杆线虫种系中的 DNA DSB 。种系与 DAPI 共同标记,以便轻松评估 DNA DSB。这种方法允许定性或定量测量 DNA DSB。

图文摘要:

C. elegans种系的 TUNEL 标记示意图。


[背景] 许多压力,无论是核压力还是其他压力,都可能导致秀丽隐杆线虫的种系表型,包括减少育雏大小或完全不育。当试图了解导致种系缺陷的机制时,有必要研究可能导致细胞凋亡的 DNA 双链断裂增加的可能性。尽管评估生殖细胞凋亡的技术已经成熟[例如,分析细胞尸体; CED-3::GFP转基因(Chen et al., 2016)]和DNA损伤[例如,LacZ报告基因(Pontier and Tijsterman, 2009);在线虫种系中的 DSB 修复标记(例如 RAD-51 和 RPA-1 (Bae et al., 2020; Hinman et al., 2021))的免疫标记,在遗传连锁的情况下包含遗传编码的报告基因可能是复杂的或不可能的。因此,生化方法可以为 DNA DSB 的评估提供更大的灵活性。如此处所述,TUNEL 标记可以识别几乎所有秀丽隐杆线虫遗传背景中的 DNA DSB(不包括那些没有性腺的)。这种方法不仅消除了生成包含报告基因的新突变菌株的需要,而且允许并行制备和分析多个样品。该协议是对所使用的协议的改编,Parusel (2006)并且与 Kropp et al. (2021).

关键字:秀丽隐杆线虫, 种系, TUNEL, 双链断裂



材料和试剂


25 × 75 mm Superfrost Plus Slides( Diagger ,目录号:EF15978Z),在室温下储存
25 × 25-1显微镜盖玻片( Fisherbrand ,目录号:12-542-C),室温储存
23 G 1¼ Precision Glide Needle(BD Bioscience,目录号:305120),室温储存
KimWipes ( Kimtech Science,Kimberly-Clark Professional,目录号:05511),在室温下储存
M9 Buffer(IPM Scientific,目录号: 11006-517),室温储存
10 × PBS(KD Medical,目录号: RGF-3210),室温储存
聚-L-赖氨酸溶液(Sigma Life Science,目录号:P8920-100mL),室温储存
多聚甲醛粉末(Sigma-Aldrich,目录号:158127),储存于 4°C
Triton X-100(Sigma Life Science,目录号:X100-100mL),室温储存
柠檬酸钠三水合物粉末(Sigma-Aldrich,目录号:C8532),室温储存
TUNEL Assay Kit – FITC(Abcam,目录号:ab66108),储存于 4°C 和 -20°C
DAPI粉末(Sigma-Aldrich,目录号:D9542),4°C储存
Vectashield (Vector Laboratories, Inc ,目录号:H-1000),在 4°C 下储存
显微镜用浸油( Cargille ,目录号:16482),室温储存


库存解决方案
4% 多聚甲醛 (PFA) 溶液(参见食谱)。
PTX 溶液(见配方)
100 mM 柠檬酸钠缓冲液(参见配方)
DAPI 解决方案(见配方)
TUNEL 检测试剂盒中的 TUNEL 反应混合物(参见配方)
1 M柠檬酸钠(见食谱)
设备


SMZ645立体显微镜(尼康,型号:SMZ645-L)
数字加热块(VWR Scientific Products,目录号:13259-050)
转盘共聚焦显微镜
尼康 Eclipse Ti2-E 倒置显微镜
横河CSUZ-1转盘
光度计 Prime95B 相机
Nikon Plan Flour 40 × /1.3 油浸物镜


软件


Elements Advanced Research(尼康, https://www.microscope.healthcare.nikon.com/products/software/nis-elements/nis-elements-advanced-research )


程序


工作试剂制备
准备聚-L-赖氨酸涂层载玻片
添加 3–5 μ L Poly-L-赖氨酸到Superfrost Plus 载玻片的中心。
将聚-L-赖氨酸溶液的液滴扩散到直径约 1 厘米的均匀层中,移液管尖端的一侧。
在室温 (RT) 下干燥过夜。载玻片干燥后可在 4°C 下保存长达一个月。
在手术前让幻灯片进入 RT。
准备 4% 多聚甲醛 (PFA) 溶液(见食谱)。
准备 PTX 溶液(见配方)。
准备 100 mM 柠檬酸钠缓冲液(参见食谱)。
准备 DAPI 解决方案(参见食谱) 。多余的溶液可在4°C 下避光保存长达 1 个月。




图 1. 从盘子到载玻片挑选动物。
(A) 使用铂丝拾取器,将动物从培养板中挑选到含有 M9 缓冲液的手表玻璃上。 (B) 使用扫掠动作,轻轻地将动物从拾取器中取出,放入手表玻璃中的 M9 缓冲器中。 (C) 使用扫动动作,轻轻地将单个动物从手表玻璃上提起,并将它们转移到含有一滴 M9 缓冲液的幻灯片上。 (D) 在聚-L-赖氨酸干燥床上的 M9 缓冲液滴中包含单个动物的幻灯片的代表性示意图。表示不是按比例的。


样品制备
从线虫(L4 或成人)的同步种群中,手动将个体动物挑选到含有 M9 溶液的手表玻璃中,以去除多余的细菌(图 1A 和 1B)。
将 10 μ L的 M9 溶液移至载玻片的聚-L-赖氨酸涂层区域。
单独挑选线虫进入幻灯片上的 M9 液滴(图 1C和1D)。总共向液滴中添加约 10 只动物。
注意:从手表玻璃中挑选蠕虫时,请用您的镐轻轻地将它们单独舀起。 M9 的表面张力将允许您拾取单个蠕虫。轻轻扫过载玻片上的 M9 液滴,将它们从拾取器中取出。
在幻灯片上,解剖动物的性腺
使用针的边缘,从幻灯片上的线虫切割头部和/或尾部。性腺会自动挤出并粘在聚-L-赖氨酸涂层上(图 2)。
笔记: 
1)有些人喜欢使用两根针和一个剪刀动作,尽管我们发现这是不必要的。
2) 加入麻醉剂如 1mM 左旋咪唑可能会有所帮助。
3) 视频协议可在https://www.jove.com/v/20131/c-elegans-gonad-extrusion-a-rapid-dissection-technique找到 (Gervaise and Arur, 2016).
快速工作,因为所有性腺都应在两分钟内解剖。




秀丽隐杆线虫的性腺解剖。
具有 23 G 1¼ 针的线虫进行解剖显微镜的代表性图像。针尖靠近咽和肠的交界处(也由浅色变为深色表示)。这是应该做切口的地方。 (B) 通过解剖显微镜观察的挤出性腺的代表性图像。肠子也会挤出来。请注意,在这种情况下,头部并未完全移除。 (C) 已突出的第一天成年性腺的增强图像(来自 B),区域已标记。 MZ:有丝分裂区; TZ:过渡区。比例尺(A 和 B):500 µm; (C) 100 µm。


用 PFA 修复样本
KimWipe的边缘吸走多余的 M9 溶液(图 3)。
吸取 25 μ L的 4% PFA 到样品上。轻轻添加盖玻片以防止蒸发。
在 RT 孵育 20 分钟。
小心地取下盖玻片。用吸管小心地取出 PFA并放入适当的容器中。
笔记:
1) 可能需要使用剃须刀片在盖玻片和载玻片之间形成分离。如果使用,请确保不要损坏样品。
2) PFA 是一种危险废物。根据您所在机构的指导方针处理它。在 PFA 固定后立即进行的所有 PTX 洗涤都应假定含有 PFA 并与 PFA 一起处理。
25–50 μL PTX 溶液清洗样品3 次,每次 3 分钟。使用与 PFA 相同的移液技术,以避免损坏样品。在与 PFA 相同的容器中处理 PTX洗涤液。




图 3. 固定、清洗和染色挤出性腺的示意图。
动物将被包含在一滴缓冲液中。在每个步骤结束时移取或吸走(使用KimWipe )缓冲液。在每一步开始时用吸管添加缓冲液。为避免破坏样品,请从缓冲液滴边缘移液或吸管,如红色椭圆中的区域所示。如有必要,倾斜滑块以使吸液更容易。表示不按比例。


在准备好的柠檬酸钠缓冲液中孵育样品以改善抗原修复
吸取 25 μ L的 10 mM柠檬酸钠溶液并盖上盖玻片。
将幻灯片放在预热到 65 °C 的热块上。孵育 20 分钟。
小心地取下盖玻片。用KimWipe的边缘吸走柠檬酸钠溶液。
如步骤 5e 所示,用 PTX 洗涤 3 次。 PTX 清洗液此时不需要特殊处理。
在 TUNEL 反应混合物中孵育样品
从 TUNEL 检测试剂盒中制备 TUNEL 反应混合物(参见配方)。
吸取 51 μ L反应混合物到样品上。添加盖玻片以防止蒸发。
在 37 °C的潮湿室中孵育60 分钟。从这一点向前保护幻灯片免受光线照射。
注意:任何带有湿纸巾的塑料容器都足以制作一个潮湿的房间。如果没有自然的方法来防止载玻片直接放在纸巾上,则加入一些支撑载玻片的东西(例如两个平行的血清移液管)效果很好。
如步骤 5e 所示,用 PTX 洗涤 3 次。 PTX 清洗液此时不需要特殊处理。
DAPI 联合标记
KimWipe边缘的 Wick away PTX 解决方案。
中加入 25 μ L DAPI 溶液并在避光室温下孵育 5 分钟
如步骤 5e 所示,用 PTX 洗涤 3 次。 PTX 清洗液此时不需要特殊处理。
安装和盖滑
KimWipe的边缘吸走任何剩余的 PTX 解决方案。
将一滴Vectashield直接添加到样品中。
轻轻地将干净的盖玻片放到样品上。为避免气泡,先降低一个边缘,然后慢慢降低盖玻片的其余部分。
注意:如果存在气泡,请用牙签轻轻按压气泡,将其移至盖玻片边缘。
用KimWipe吸走多余的Vectashield 。用指甲油密封盖玻璃。
样品可以立即或第二天成像。第二天成像往往会产生更好的结果。如果等待图像,请将幻灯片存放在 4 °C。




与 DAPI 和 TUNEL 共同标记的野生型 L4线虫性腺的代表性最大强度投影。
DAPI 染色的细胞核标记为蓝色,TUNEL 阳性细胞核标记为绿色。由于这是 L4 性腺,因此不存在卵母细胞。基于核 (DAPI) 结构可辨别性腺的不同区域。由于减数分裂期间的交叉形成(双倍体),细胞核将用 TUNEL 标记。在其他系统中报道了用 TUNEL 对有丝分裂核进行人工标记,包括人体组织切片(Labat-Moleur et al., 1997)和果蝇卵巢(Qi and Calvi, 2016)。复制过程中的 DNA 缺口可能是原因(Mets and Meyer, 2009)。中间粗线期区域的明显苍白标记是将两个 z 堆栈拼接成单个图像的伪影。比例尺:100 μ m 。


共聚焦成像
准备
确保所有适当的激光器(405 nm 和 488 nm)和光源都打开并加热。将 405 nm 激光设置为 35% 功率和 100 ms曝光。将 488 nm 激光设置为 25.5% 功率和 100 ms曝光。
将载玻片安装到显微镜台上。
如果使用油浸物镜,在盖玻片上加一小滴油。
找到所需的样本。
图像采集
将 z 切片厚度设置为 0.3 微米。 _
定义 z 堆栈以包含整个种系直径。
捕获 z 堆栈。
如有必要,捕获第二个 z 堆栈以对整个生殖系进行成像。


数据分析


数据分析应根据最终最终用途由实验者自行决定。我们定性评估了 TUNEL 标记,但可以通过确定 TUNEL 阳性细胞核的百分比作为总细胞核 ( (# TUNEL+)/(# DAPI+)) 的函数或性腺特定区域(例如粗线期)内 TUNEL 阳性细胞核的百分比来产生定量测量。比较野生型和突变型样品之间 TUNEL 阳性细胞核的百分比是合适的。


笔记 


增加 DSB 发生率的阳性对照是将秀丽隐杆线虫暴露于紫外线下。我们通过将带有线虫的盘子放在标准紫外线灯箱上 20 分钟来实现这一点。平板上接种了 OP50 细菌,尽管我们不认为这是明确必要的。


食谱


所有的缓冲液都是购买的。孵育和洗涤缓冲液的制备如下所示。
4% 多聚甲醛 (PFA) 溶液
在 H 2 O 中,来自 16% 的库存。用新鲜的。可能需要加热。储存在 4 °C 。
PTX 解决方案
10 × PBS 库存和 Triton X-100。 1 × PBS + 0.4% Triton X-100。用新鲜的。
100 mM 柠檬酸钠缓冲液
1 M 库存 + 0.1% Triton X-100。用新鲜的。
DAPI 解决方案
10 μ g /mL 在 H 2 O中。
TUNEL 检测试剂盒中的 TUNEL 反应混合物
10 μL Reaction Buffer 、 0.75 μL TdT Enzyme 、 8 μL FITC - dUTP 、 32.25 μL ddH 2 O。
1 M 柠檬酸钠
在 H 2 O 中。可能需要加热。在室温下储存。


致谢


这项工作得到了美国国立卫生研究院、国家糖尿病、消化和肾脏疾病研究所 (AG) 的校内研究计划的支持。资助者在研究设计、数据收集和分析、发表决定或手稿准备方面没有任何作用。该协议是对所使用的协议的改编,Parusel (2006)并且与 Kropp et al. (2021).


利益争夺


作者声明没有竞争利益。


参考


Bae, W., Park, JH, Lee, MH, Park, HW 和 Koo, HS (2020)。秀丽隐杆线虫的 exo-1 和 polq-1 突变抑制了对与 PARG 缺乏相关的 DNA 双链断裂的超敏反应。 FEBS J 287(6):1101-1115。
Chen, X., Wang, Y., Chen, YZ, Harry, BL, Nakagawa, A., Lee, ES, Guo, H. 和Xue , D. (2016)。秀丽隐杆线虫核膜蛋白 NPP-14对 CED-3 半胱天冬酶定位和激活的调节。 Nat 结构分子生物学23(11):958-964。
Gervaise , AL 和Arur , S. (2016)。秀丽隐杆线虫种系中活性 ERK 的时空分析。 J Vis Exp ( 117):54901。
Hinman, AW, Yeh, HY, Roelens , B., Yamaya , K., Woglar , A., Bourbon, HG, Chi, P. 和 Villeneuve, AM (2021)。秀丽隐杆线虫DSB-3 揭示了促进减数分裂双链断裂的蛋白质复合物之间的保守性和差异性。 Proc Natl Acad 美国科学118(33):e2109306118。
Kropp, PA, Wu, J., Reidy, M., Shrestha, S., Rhodehouse , K., Rogers, P., Sack, MN 和 Golden, A. (2021)。以秀丽隐杆线虫为模型的多发性线粒体功能障碍综合征 1 致病突变诱导的等位基因特异性线粒体应激。 公共科学图书馆基因17(8):e1009771 。
Labat-Moleur , F., Guillermet , C., Lorimier , P., Robert, C., Lantuejoul , S., Brambilla, E. 和Negoescu , A. (1998)。组织切片中的 TUNEL 凋亡细胞检测:关键评估和改进。 组织化学杂志 细胞化学46(3):327-334。
大都会,DG 和 Meyer,BJ(2009 年)。凝聚素通过控制染色体结构来调节减数分裂 DNA 断裂分布,从而调节交叉频率。 单元格139(1):73-86。
康涅狄格州帕鲁塞尔(2006)。 URI-1 是秀丽隐杆线虫DNA 稳定性所必需的。 发展133(4):621-629。
Pontier , DB 和Tijsterman , M. (2009)。一个强大的双链断裂修复通路网络控制着秀丽隐杆线虫发育过程中的基因组完整性。 电流 生物学19(16):1384-1388。
Qi, S. 和Calvi , BR (2016)。不同的细胞周期修饰在不同的步骤抑制细胞凋亡,而与果蝇的发育信号无关。 分子生物学细胞27(12):1885-1897。




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引用:Kropp, P. A., Rhodehouse, K. and Golden, A. (2022). TUNEL Labeling to Detect Double-stranded DNA Breaks in Caenorhabditis elegans Gonads. Bio-protocol 12(6): e4351. DOI: 10.21769/BioProtoc.4351.
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