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Determination of DNA Damage in the Retina Photoreceptors of Drosophila
果蝇视网膜光感受器DNA损伤测定   

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Molecular Neurobiology
Sep 2017

Abstract

The retina is sensitive for light damages, because of direct light exposure, especially intense blue and UV light, which increase level of ROS and other toxic phototransduction products in photoreceptor cells. In our previous work (Damulewicz et al., 2017a and 2017b), we used 8-oxo-deoxyguanosine (8-OHdG) as a marker for oxidative stress to investigate the role of heme oxygenase in DNA protection against UV light. In this protocol, we showed how to determine the level of DNA damages in the retina using immunohistochemical staining.

Keywords: Oxidative stress (氧化应激), 8-Oxo-deoxyguanosine (8-氧代脱氧鸟苷), UV light (紫外线), Retina degeneration (视网膜变性), DNA damage (DNA损伤)

Background

Deoxyribonucleic acid (DNA) is an essential molecule for all living organisms. It contains genetic code with instruction for proteins and other molecules necessary for structure and metabolism of an organism. DNA is composed of two strands of nucleotides. Each nucleotide is built of one of four types of nucleobases (cytosine, guanine, adenine and thymine), deoxyribose and phosphate group. Physical and chemical changes in DNA structure are classified as DNA damages. It includes one or two strands of DNA breaks, missing nucleotide or chemical changes of nucleobases. Damages can be induced by environmental factors, like chemicals or UV light exposure, or in result of metabolic processes, which produce reactive oxygen species, reactive nitrogen species, reactive carbonyl species and alkylating agents (De Bont and van Larebeke, 2004).

One of the most common DNA damage is 8-oxo-deoxyguanosine (8-OHdG), which is a product of DNA oxidation by reactive oxygen species and it serves as a marker of oxidative stress (De Souza-Pinto et al., 2001; Swenberg et al., 2011; Valavanidis et al., 2013). 8-OHdG enhances the risk of transversion mutation, because it has ability to pair with adenine and cytosine bases (Maki and Sekiguchi, 1992). In effect it plays a role in mutagenesis, cancerogenesis and aging (De Bont and van Larebeke, 2004; Kohen and Nyska, 2002). Higher 8-OHdG level was described in breast, renal and gastric tumors (Lee et al., 1998; Musarrat et al., 1996; Okamoto et al., 1994).

There are several methods to determine 8-OHdG level in a specific tissue, like immunohistochemistry (De Carvalho et al., 2013), Western blot (Kannan et al., 2006), standard HPLC (Herrero and Barja, 1999), liquid chromatography nanoelectrospray-tandem mass spectrometry (Ma et al., 2016). Here, we present a detailed protocol for DNA damage determination in the retina of fruit fly, Drosophila melanogaster using immunohistochemistry method (Damulewicz et al., 2017a and 2017b).

Materials and Reagents

  1. Filter paper (Fisher Scientific, catalog number: 09-790-14D )
  2. Cover glasses (Menzel-Glaser)
  3. Microscope glasses (Superfrost, Menzel-Glaser)
  4. Small plastic holders (i.e., Eppendorf tube lids)
  5. Tissue-Tek (SAKURA, catalog number: 4583 )
  6. Drosophila melanogaster (wild-type CantonS, 5-7 days old males)
    Note: Local ethical standards need to be followed.
  7. 6% glucose in water (commercial)
  8. Protoporphyrin IX (SnPP IX) (Frontier Scientific, catalog number: P562-9 )
  9. Hemin chloride (Merck, Calbiochem, catalog number: 3741 )
  10. CO2
  11. Schneider’s medium (Sigma-Aldrich, catalog number: S9895 )
  12. Etoposide (Sigma-Aldrich, catalog number: E1383 )
  13. 4% paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )
  14. Sucrose (commercial)
  15. Liquid nitrogen
  16. Normal Goat Serum (NGS) (Sigma-Aldrich, catalog number: G9023 )
  17. Bovine serum albumin (BSA)
  18. Mouse anti-8-hydroxyguanosine primary antibodies (Acris, catalog number: AM03160 )
  19. HRP/DAB (ABC) kit (Abcam, catalog number: ab64264 )
  20. Glycerol (MP Biomedicals)
  21. Vectashield with DAPI (Vector Laboratories, catalog number: H-1200 )
  22. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  23. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  24. Sodium phosphate monobasic monohydrate (NaH2PO4·H2O) (Sigma-Aldrich, catalog number: S3522 )
  25. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
  26. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: H1758 )
  27. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S7907 )
  28. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  29. Cornmeal (Glutenex)
  30. Agar (Bioshop)
  31. Artificial honey (Vortumnus)
  32. Yeast (Paneo)
  33. Methyl-4-hydroxybenzoate (anti-fungal chemical, Sigma-Aldrich, catalog number: H3647 )
  34. 90% EtOH
  35. Phosphate buffered saline (PBS) (see Recipes)
  36. Phosphate buffer with Triton (PBT) (see Recipes)
  37. Standard medium for flies (see Recipes)

Equipment

  1. Forceps (Dumont No. 5)
  2. UV transilluminator (Vilbert Lourmat, model: UVECX26 )
  3. White light transilluminator (Vilbert Lourmat, model: TFX53WL )
  4. Cryostat (Leica, model: CM1850 UV )
  5. Light microscope (Carl Zeiss, model: Axioskop 50 )
  6. Camera (Canon, model: G10 )

Software

  1. ImageJ software

Procedure

  1. Take 30 males for every group (experimental and control).
    Notes: You can use different control groups, according to your experiment:
    1. Control for the staining conditions: positive (incubated with etoposide) and negative (without primary antibodies).
    2. Control for UV light effect–flies unexposed to light (kept in constant darkness).
    3. Control for feeding effect–flies fed with glucose only.
  2. Keep flies for 5 days in constant darkness (DD) on a standard medium.
  3. Place flies for 6 h into an empty vial, with no access to food, but with wet filter paper to provide them water ad libitum. Keep them in DD conditions.
  4. Apply selected chemicals diluted in 6% glucose to a filter paper for 6 h or 6% glucose only for control groups. In this experiment, we used heme oxygenase inhibitor (100 µM protoporphyrin IX–SnPP IX) and activator (100 µM hemin chloride).
    Note: Avoid leaving big droplets of a liquid at the bottom of vial–flies can be trapped in liquid and drown.
  5. Use CO2 to anesthetize one of the control groups.
  6. Decapitate one of the control groups and put heads for 4 h in 500 µl Schneider’s medium supplemented with 20 µM etoposide at room temperature.
  7. Expose experimental flies and second control group (fed with glucose only) to 1 h of UV light pulse (100 lux) and then to 3 h of intensive white light (1,500 lux) (Figure 1).
    Note: Be sure that flies have access to water during the experiment (wet filter paper).


    Figure 1. UV/white light exposure. Vial (orientated upside down) with flies is placed on the transilluminator. Filter paper attached to the vial provide water ad libitum.

  8. Keep one of control group continuously in constant darkness until decapitation.
  9. Use CO2 to anesthetize flies.
  10. Decapitate the anesthetized flies (all experimental and control) in a drop of 4% PFA.
  11. Fix all heads in 500 µl 4% PFA for 4 h at 4 °C.
  12. Remove PFA using a pipette and wash heads 2 times each for 10 min at room temperature (RT) in 500 µl PBS (see Recipes).
  13. Cryoprotect material by washing for 10 min in 500 µl of 12.5% sucrose and then overnight in 500 µl of 25% sucrose (Figure 2).


    Figure 2. Fixation and cryoprotection of material. Flies heads are fixed in 4% PFA, then washed in PBS and cryoprotected in 12.5% and 25% sucrose.

  14. Put a droplet of Tissue-Tek to small plastic holders, embed heads into Tissue-Tek, put them into the bottom and organize in rows (Figure 3).


    Figure 3. Sample preparation for cryostat. Small plastic holder is filled with TissueTek, then heads are put into the bottom in rows and cut (frontal sections). Sections are collected on microscope glass. 

  15. Freeze material in liquid nitrogen for few seconds.
  16. Remove frozen sample from the holder.
  17. Use a cryostat to cut 20 µm thick sections.
  18. Dry the cryosections for 30 min at room temperature.
  19. Keep the sections frozen for long-term storage at -20 °C or use them immediately for immunodetection at room temperature.
  20. Rehydrate material by incubating with 500 µl PBS for 30 min.
    Note: Use PBS, PBT at room temperature.
  21. Wash sections for 10 min in 200 µl 0.5% PBT (see Recipes).
  22. Wash sections for 5 min in 200 µl 2% PBT.
  23. Wash sections 3 times for 5 min each in 200 µl 0.5% PBT.
  24. Block unspecific binding sites by the incubation with 200 µl 5% NGS in BSA for 45 min.
  25. Apply anti-8-hydroxyguanosine primary antibodies (1:500, in 0.5% BSA) and incubate overnight at 4 °C.
  26. Keep one section as a negative control and do not apply primary antibodies.
  27. Use HRP/DAB (ABC) detection kit according to the manufacturer’s protocol. In this step secondary antibodies are used (they are provided in the kit).
  28. Close sections with glycerol and cover glasses.
  29. Take the retina pictures under a light microscope using a microscope camera.
    Note: To visualize cell nuclei in the retina keep one section unstained with anti-8OHdG. Rehydrate material and close with fluorescence medium–Vectashield with DAPI. Use a fluorescent or confocal microscope to take pictures.

Data analysis

  1. Analyze DNA damages as Mean Grey Values using ImageJ software. Select area on the top of retina (where nuclei are located, see Figure 5) and measure Mean Grey Value (the range between 0-255). Mean Grey Value is the sum of the grey values of all pixels in the area divided by the number of pixels within selection. Collect three measurements from one picture, use the average as a mean for this picture, collect at least 20 means (different individuals) (Table 1). Copy your data to Excel file and compare means between experimental and control groups.
    Note: The retina photoreceptors nuclei are located in the distal part of the retina (see Figures 4 and 5, nuclei are stained with DAPI), measure Mean Grey Value in this area only. Use the same microscope and camera settings for all groups (experimental and control).


    Figure 4. Controls used for DNA damage determination. A. Negative control–without primary antibodies. B. Positive control–etoposide treatment. Scale bars = 20 µm.


    Figure 5. Localization of cell nuclei in the retina. A. DAPI staining–cell nuclei are visualized. B. Retina immunostained with anti-8-hydroxyguanosine, black square shows an analyzed area. Scale bar = 20 µm.

    Table 1. Sample triplicate measurements for experimental and control flies

Recipes

  1. Phosphate buffer (PBS, 1x)
    8 g NaCl
    0.2 g KCl
    1.44 g Na2HPO4
    0.24 g KH2PO4
    800 ml distilled H2O
    Adjust pH to 7.4 with HCl
    Adjust volume to 1 L with distilled H2O
  2. Phosphate buffer with Triton (PBT, 1 L)
    0.4363 g NaH2PO4·H2O
    0.9705 g Na2HPO4
    7.953 g NaCl
    5 ml Triton X-100 for 0.5% PBT
    Distilled water up to 1 L
    Note: PBS and PBT can be stored for a few weeks in the fridge.
  3. Standard medium for flies
    120 g cornmeal (Glutenex)
    10 g agar (Bioshop)
    140 ml artificial honey (Vortumnus)
    10 g yeast (Paneo)
    2 L of water
    Boil 30 min
    Add 3 g methyl-4-hydroxybenzoate (anti-fungal chemical, Sigma-Aldrich) in 15 ml 90% EtOH

Acknowledgments

The study was supported by the Polish National Science Centre (NCN) grant No. UMO-2012/07/B/NZ3/02908 to EP. The protocol was adapted according to our previous papers: Damulewicz, M., Loboda, A., Jozkowicz, A., Dulak, J. and Pyza, E. (2017). Interactions between the circadian clock and heme oxygenase in the retina of Drosophila melanogaster. Mol. Neurobiol 54(7): 4593-4962. Damulewicz, M., Loboda, A., Jozkowicz, A., Dulak, J. and Pyza, E. (2017). Haeme oxygenase protects against UV light DNA damages in the retina in clock-dependent manner. Sci Rep 7(1): 5197. The authors declare that they have no conflicts or competing interests.

References

  1. Damulewicz, M., Loboda, A., Jozkowicz, A., Dulak, J. and Pyza, E. (2017a). Interactions between the circadian clock and heme oxygenase in the retina of Drosophila melanogaster. Mol Neurobiol 54(7): 4593-4962.
  2. Damulewicz, M., Loboda, A., Jozkowicz, A., Dulak, J. and Pyza, E. (2017b). Haeme oxygenase protects against UV light DNA damages in the retina in clock-dependent manner. Sci Rep 7(1): 5197.
  3. De Bont, R. and van Larebeke, N. (2004). Endogenous DNA damage in humans: a review of quantitative data. Mutagenesis 19(3): 169-185.
  4. de Carvalho, L. F., Abrao, M. S., Biscotti, C., Sharma, R., Agarwal, A. and Falcone, T. (2013). Mapping histological levels of 8-hydroxy-2'-deoxyguanosine in female reproductive organs. J Mol Histol 44(1): 111-116.
  5. de Souza-Pinto, N. C., Eide, L., Hogue, B. A., Thybo, T., Stevnsner, T., Seeberg, E., Klungland, A. and Bohr, V. A. (2001). Repair of 8-oxodeoxyguanosine lesions in mitochondrial dna depends on the oxoguanine dna glycosylase (OGG1) gene and 8-oxoguanine accumulates in the mitochondrial dna of OGG1-defective mice. Cancer Res 61(14): 5378-5381.
  6. Herrero, A. and Barja, G. (1999). 8-oxo-deoxyguanosine levels in heart and brain mitochondrial and nuclear DNA of two mammals and three birds in relation to their different rates of aging. Aging (Milano) 11(5): 294-300.
  7. Kannan, S., Pang, H., Foster, D. C., Rao, Z. and Wu, M. (2006). Human 8-oxoguanine DNA glycosylase increases resistance to hyperoxic cytotoxicity in lung epithelial cells and involvement with altered MAPK activity. Cell Death Differ 13(2): 311-323.
  8. Kohen, R. and Nyska, A. (2002). Oxidation of biological systems: oxidative stress phenomena, antioxidants, redox reactions, and methods for their quantification. Toxicol Pathol 30(6): 620-650.
  9. Lee, B. M., Jang, J. J. and Kim, H. S. (1998). Benzo[a]pyrene diol-epoxide-I-DNA and oxidative DNA adducts associated with gastric adenocarcinoma. Cancer Lett 125(1-2): 61-68.
  10. Ma, B., Jing, M., Villalta, P. W., Kapphahn, R. J., Montezuma, S. R., Ferrington, D. A. and Stepanov, I. (2016). Simultaneous determination of 8-oxo-2'-deoxyguanosine and 8-oxo-2'-deoxyadenosine in human retinal DNA by liquid chromatography nanoelectrospray-tandem mass spectrometry. Sci Rep 6: 22375.
  11. Maki, H. and Sekiguchi, M. (1992). MutT protein specifically hydrolyses a potent mutagenic substrate for DNA synthesis. Nature 355(6357): 273-275.
  12. Musarrat, J., Arezina-Wilson, J. and Wani, A. A. (1996). Prognostic and aetiological relevance of 8-hydroxyguanosine in human breast carcinogenesis. Eur J Cancer 32A(7): 1209-1214.
  13. Okamoto, K., Toyokuni, S., Uchida, K., Ogawa, O., Takenewa, J., Kakehi, Y., et al. (1994). Formation of 8-hydroxy-2’-deoxyguanosine and 4-hydroxy-2-nonenal-modified proteins in human renal-cell carcinoma. Int J Cancer 58: 825-829.
  14. Swenberg, J. A., Lu, K., Moeller, B. C., Gao, L., Upton, P. B., Nakamura, J. and Starr, T. B. (2011). Endogenous versus exogenous DNA adducts: their role in carcinogenesis, epidemiology, and risk assessment. Toxicol Sci 120 Suppl 1: S130-145.
  15. Valavanidis, A., Vlachogianni, T., Fiotakis, K. and Loridas, S. (2013). Pulmonary oxidative stress, inflammation and cancer: respirable particulate matter, fibrous dusts and ozone as major causes of lung carcinogenesis through reactive oxygen species mechanisms. Int J Environ Res Public Health 10(9): 3886-3907.

简介

视网膜对光损伤敏感,因为直接光照,尤其是强烈的蓝光和紫外光,这增加了光感受器细胞中ROS和其它有毒光转导产物的水平。 在我们以前的工作(Damulewicz等人,2017a和2017b)中,我们使用8-氧代 - 脱氧鸟苷(8-OHdG)作为氧化应激的标记来研究血红素加氧酶在DNA保护中的作用 防紫外线。 在这个协议中,我们展示了如何使用免疫组织化学染色来确定视网膜中DNA损伤的水平。

【背景】脱氧核糖核酸(DNA)是所有生物体的必需分子。它含有遗传密码,其中含有对生物体结构和代谢所必需的蛋白质和其他分子的说明。 DNA由两条核苷酸链组成。每种核苷酸是四种类型的核苷碱基(胞嘧啶,鸟嘌呤,腺嘌呤和胸腺嘧啶),脱氧核糖和磷酸酯基团中的一种。 DNA结构中的物理和化学变化被归类为DNA损伤。它包括一个或两个DNA断裂链,缺少核碱基的核苷酸或化学变化。损坏可能是由于环境因素诱导,如化学物质或UV光暴露,或在代谢过程,其产生活性氧,活性氮物质,反应性羰基物质和烷化剂(德邦特和van Larebeke,2004)的结果。

其中最常见的DNA损伤是-8-氧代脱氧鸟苷(8-OHdG的),所有这些是由活性氧物种的DNA氧化的产物,它作为氧化应激(德索萨-Pinto的等的标记。 ,2001,Swenberg等人,2011; Valavanidis等人,2013)。 8-OHdG由于其对腺嘌呤和胞嘧啶碱基的能力而增加了颠换突变的风险(Maki和Sekiguchi,1992)。实际上,它在诱变,癌变和衰老中发挥作用(Bont和van Larebeke,2004,Kohen和Nyska,2002)。较高的8-OHdG水平,这在描述乳房,肾和胃肿瘤(李等人,1998年Musarrat 等人,1996;冈本等。 ,1994)。

有几种方法来确定性矿的8-OHdG水平在特定组织,如免疫组织化学(德卡瓦略等人,2013年),Western印迹的(Kannan 等人,2006年),标准HPLC(Herrero和Barja,1999),液相色谱纳电喷雾串联质谱法(Mae等人,2016)。这里,我们提出对DNA损伤测定的详细协议在果蝇的视网膜,果蝇使用免疫组织化学法(Damulewicz 等人,2017A和2017b)。

关键字:氧化应激, 8-氧代脱氧鸟苷, 紫外线, 视网膜变性, DNA损伤

材料和试剂

  1. 滤纸(Fisher Scientific,目录号:09-790-14D)。
  2. 盖玻璃(Menzel-Glaser)
  3. 显微镜眼镜(Superfrost,Menzel-Glaser)
  4. 小塑料支架(即Eppendorf管盖)
  5. 组织Tek(SAKURA,目录号:4583)
  6. 果蝇(野生型,5-7天龄的男性)
    注意:必须遵守当地的道德标准。
  7. 6%葡萄糖在水中(商业)
  8. 原卟啉IX(SnPP IX)(Frontier Scientific,目录号:P562-9)。
  9. 氯化血红素(Merck,Calbiochem,目录号:3741)
  10. CO 2
  11. 施耐德培养基(Sigma-Aldrich,目录号:S9895)
  12. 依托泊苷(Sigma-Aldrich,目录号:E1383)。
  13. 4%多聚甲醛(PFA)(Sigma-Aldrich,目录号:P6148)。
  14. 蔗糖(商业)
  15. 液氮
  16. 正常山羊血清(NGS)(Sigma-Aldrich,目录号:G9023)
  17. 牛血清白蛋白(BSA)
  18. 小鼠抗8-羟基鸟苷一级抗体(Acris,目录号:AM03160)。
  19. HRP / DAB(ABC)试剂盒(Abcam,目录号:ab64264)
  20. 甘油(MP生物医学)
  21. Vectashield与DAPI(矢量实验室,目录号:H-1200)。
  22. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  23. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  24. 磷酸二氢钠一水合物(NaH2)PO4•2H2O(Sigma-Aldrich,目录号:S3522)
  25. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P5655)。
  26. 盐酸(HCl)(Sigma-Aldrich,目录号:H1758)
  27. 磷酸二氢钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S7907)
  28. Triton X-100(Sigma-Aldrich,目录号:T8787)
  29. 玉米面(Glutenex)
  30. 琼脂(Bioshop)
  31. 人造蜂蜜(Vortum)
  32. 酵母(Paneo)
  33. 4-羟基苯甲酸甲酯(抗真菌化学品,Sigma-Aldrich,目录号:H3647)。
  34. 90%EtOH
  35. 磷酸盐缓冲盐水(PBS)(见食谱)
  36. 用Triton磷酸盐缓冲液(PBT)(见食谱)。
  37. 苍蝇的标准培养基(见食谱)

设备

  1. 镊子(杜蒙特5号)
  2. 紫外透射仪(Vilbert Lourmat,型号:UVECX26)
  3. 白光透照器(Vilbert Lourmat,型号:TFX53WL)
  4. 低温恒温器(徕卡,型号:CM1850紫外线)
  5. 光学显微镜(卡尔蔡司,型号:Axioskop 50)
  6. 相机(佳能,型号:G10)

软件

  1. ImageJ软件

程序


  1. 每组30次(实验和控制) 注意:根据您的实验,您可以使用不同的控制组:
    1. 对染色条件的控制:阳性(与依托泊苷孵育)和阴性(不含第一抗体)。
    2. 控制紫外线照射效果,不受光照影响(保持恒定黑暗)。
  2. 将标准培养基中的苍蝇保持恒定黑暗(DD)5天。
  3. 将苍蝇放置6小时到一个空的小瓶中,没有食物,但用湿滤纸给他们提供“随意”饮用水。保持他们在DD条件。
  4. 将选定的稀释在6%葡萄糖中的化学物质仅用于对照组,6%或6%葡萄糖。在这个实验中,我们使用了乳清加氧酶抑制剂(100μM原卟啉IX-SnPP IX)和活化剂(100μM氯化血红素)。
    注意:避免小瓶底部的液滴泄漏,可能会被困在液体中并淹死。
  5. 使用CO 2 来麻醉其中一个对照组。
  6. 将其中一个对照组剔除,并在室温下用500μLSchneider's培养基(其补充有20μM依托泊苷)静置4h。
  7. 将实验苍蝇和第二对照组(仅用葡萄糖喂食)到1h的UV光脉冲(100lux),然后到3h的强烈的白光(1.500lux)(图1)。
    注意:确保瓷砖在实验过程中可以使用水(湿滤纸)。


    图1.紫外线/白光照射。将透明器上的苍蝇放在上面。
    过滤纸贴在小瓶上,可以自由饮水
  8. 保持对照组的一个连续黑暗直到斩首。
  9. 使用CO 2 麻醉苍蝇。

  10. 在一滴4%PFA中将麻醉的苍蝇(所有实验和对照)斩首

  11. 在500μl4%PFA中,在4°C下固定4 h
  12. 使用移液管去除PFA,并在室温(RT)下在500μlPBS中各洗2次,每次10分钟(见食谱)。
  13. 通过在500μl的12.5%蔗糖中洗涤10分钟,然后在500μl的25%蔗糖中过夜来冷冻保护材料(图2)。


    图2.固定和低温保护的材料。苍蝇头固定在4%PFA,然后在PBS中洗涤,并在12.5%和25%蔗糖中进行低温保护。

  14. 将一滴Tissue Tek放入小塑料支架中,将头部嵌入Tissue Tek,将它们放入底部并按行排列(图3)。


    图3.低温恒温器的样品制备用TissueTek填充小塑料支架,然后将头放入底部并切割(正面部分)。
    在显微镜玻璃上收集切片
  15. 在液氮中冷冻材料几秒钟。

  16. 取出冷冻样品

  17. 使用低温恒温器切割20微米厚的部分

  18. 在室温下冷冻30分钟
  19. 保持冷冻部分在-20°C长期储存或立即使用它们在室温下进行免疫检测。

  20. 用500μlPBS孵育30分钟,使材料再生 注意:在室温下使用PBS,PBT。
  21. 在200μl0.5%PBT中清洗10分钟(见食谱)。
  22. 在200μl2%PBT中清洗5分钟。

  23. 在200μl0.5%PBT中洗涤3次,每次5分钟
  24. 通过与200μl5%NGS在BSA中孵育45分钟来阻断非特异性结合位点。
  25. 应用抗8-羟基鸟苷一级抗体(1:500,在0.5%BSA中)并在4℃孵育过夜。
  26. 保留一个部分作为阴性对照,不要使用一抗。
  27. 根据制造商的协议使用HRP / DAB(ABC)检测试剂盒。在这个步骤中,使用二抗(在试剂盒中提供)。
  28. 用甘油关闭部分并盖上眼镜。

  29. 使用显微镜照相机在显微镜下拍摄视网膜图片 注:为了显现视网膜中的细胞核,使其中一个部分未被抗-80OHdG染色。用DAPI补充材料并用荧光介质-Vectashield封闭。使用荧光或共焦显微镜拍照。

数据分析

  1. 使用ImageJ软件将DNA损伤分析为平均灰度值。选择视网膜顶部的区域(原子核所在的位置,见图5),并测量平均灰度值(0-255之间的范围)。平均灰度值是该区域中所有像素的灰度值的总和。从一幅图片中收集三个测量值,用平均值作为该图片的均值,收集至少20个平均值(不同的个人)(表1)。将数据复制到实验组和控制组之间的Excel文件中。
    注:视网膜感光细胞核位于视网膜的远端部分(见图4和图5,细胞核用DAPI染色),仅测量该区域的平均灰度值。使用相同的显微镜和相机设置为所有组(实验和控制)。


    图4.用于DNA损伤测定的对照。 A.阴性对照 - 无一抗。 B.阳性对照依托泊苷治疗。比例尺= 20微米。


    图5.视网膜核的定位。 A. DAPI染色 - 细胞核被可视化。用抗8-羟基鸟苷免疫染色的视网膜,黑色方块显示分析区域。比例尺= 20微米。

    表1.实验和对照果蝇的一式三份样品
    “”

食谱

  1. 磷酸盐缓冲液(PBS,1x)
    8克NaCl
    0.2克KCl
    1.44g的Na 2 HPO 4 4 0.24克KH 2 PO 4 4 800ml蒸馏H 2 O. 用HCl调节pH值到7.4。
    用蒸馏水调节体积至1L
  2. 用Triton磷酸盐缓冲液(PBT,1 L)
    0.4363克的NaH <子> 2 PO <子> 4 •H <子> 2 0
    0.9705克Na 2 HPO 4 4 7,953克NaCl
    用于0.5%PBT的5ml Triton X-100

    蒸馏水最多1升 评级:PBS和PBT可以在冰箱里储存几周。
  3. 瓷砖的标准介质
    120克玉米面(面筋eex)
    10克琼脂(Bioshop)
    140毫升人造蜂蜜(Vortum)
    10克酵母(Paneo)
    2升水。
    煮30分钟
    加入3g 4-羟基苯甲酸甲酯(抗真菌化学品,Sigma-Aldrich)在15ml 90%EtOH

确认

该研究得到了波兰国家科学中心(NCN)的批准。 UMO-2012/07 / B / NZ3 / 02908。的协议,该协议angepasstgemäß我们以前的论文:Damulewicz,M. Loboda,A.,Jozkowicz,A.,Dulak,J。和Pyza,E。(2017)。在视网膜的昼夜节律钟和血红素加氧酶之间的相互作用的果蝇。 分子。 Neurobiol 54(7):4593-4962。 Damulewicz,M. Loboda,A.,Jozkowicz,A.,Dulak,J。和Pyza,E。(2017)。 Haeme加氧酶以时钟依赖的方式防止视网膜中的紫外线DNA损伤。科学家报告7(1):5197。作者声明他们没有冲突或利益冲突。

参考

  1. Damulewicz,M. Loboda,A.,Jozkowicz,A.,Dulak,J。和Pyza,E。(2017)。在视网膜的昼夜节律钟和血红素加氧酶之间相互作用的果蝇 Neurobiol的摩尔数 54(7):4593-4962。
  2. Damulewicz,M. Loboda,A.,Jozkowicz,A.,Dulak,J。和Pyza,E。(2017b)。 海门氧合在从属时钟的方式视网膜保护皮肤免受紫外线光的损害的DNA。 Sci Rep 7(1):5197。
  3. De Bont,R.和van Larebeke,N.(2004)。在人类中内源性DNA损害:定量数据代码评论诱变<。 / 19(3):169-185。
  4. 德卡瓦略,L. F.,Abrao,M.S.,比斯科蒂,C.夏尔马,R.阿加瓦尔,A。和Falcone的,T。(2013)。 地图在雌性生殖器官8-羟基-2'-脱氧鸟苷的组织学水平。
    J Mol Histol 44(1):111-116。
  5. 德索萨-平,N.C。,艾德,L.霍格,B. A.,Thybo,T.,Stevnsner,T.,西贝尔格,E.,Klungland,A。和玻尔,V. A.(2001)。线粒体DNA 8 oxodeoxyguanosine病变修复依赖于鸟嘌呤DNA糖基化酶(OGG1)基因和8 。-oxoguanine积聚在的缺陷OGG1小鼠代码线粒体DNA 癌症研究 61(14):。5378-5381
  6. Herrero,A。和Barja,G。(1999)。在心脏和大脑线粒体和三分之二的哺乳动物和鸟类的核DNA 8氧代脱氧鸟苷水平老龄问题(米兰) 11(5):294-300。
  7. 卡纳安,S.,庞,H.福斯特,D.C.,饶,R.和Wu,M。(2006)。 人类8-羟基鸟嘌呤DNA糖基化酶,以增加高氧的细胞毒性电阻在肺上皮细胞和参与具有改变的MAPK活性。细胞死亡不同 13(2):311-323。
  8. Kohen,R。和Nyska,A。(2002)。生物系统的氧化:氧化应激现象,抗氧化剂,氧化还原反应,并为他们的定量方法“。 / A> 毒理学病理学 30(6):. 620-650
  9. Lee,B.M.,Jang,J.J.和Kim,H.S.(1998)。 苯并[a]芘二醇环氧化物-I DNA和氧化胃腺癌相关的DNA加合物。 癌症通讯 125(1-2):61-68。
  10. 马,B.,京,M.,比利亚尔塔,P. W.,Kapphahn,R.J.,蒙特苏马,S.R.,Ferrington,D. A.和斯捷潘诺夫,I。(2016)。 -8-氧代-2'-脱氧鸟苷和8-氧代-2'-脱氧腺苷在同时测定人视网膜DNA通过液相色谱电喷雾纳米 - 串联质谱法码科学代表 6:第二万二千三百七十五
  11. Maki,H。和Sekiguchi,M。(1992)。 Mutt的蛋白特异性水解的有效诱变底物DNA的合成。 自然< 355(6357):273-275。
  12. Musarrat,J.,Arezina-Wilson,J.and Wani,A.A.(1996)。 8 Hydroxyguanosine在人乳腺癌的发生预后和病原学的相关性。 欧元J Cancer 32A(7):1209-1214。
  13. 冈本,K.,丰国,S.,内田,K。,小川,O.,Takenewa,J.,笕,Y。,等。 (1994)。的形成8-羟基-2'-脱氧鸟苷和4-羟基-2-壬烯醛修饰的蛋白质在人肾细胞癌中。 Int J Cancer 58:825-829。
  14. Swenberg,J. A.,路,K.,穆勒,B.C.,高,L.,厄普顿,P. B.,中村,J。和斯塔尔,T. B.(2011)。 内生外源性与DNA加合物:其在癌变,流行病学的作用和风险评估的代码。毒性科学 120补充1:S130-145。
  15. Valavanidis,A.,Vlachogianni,T.,Fiotakis,K。和Loridas,S。(2013)。 肺氧化应激,炎症和癌症:可吸入颗粒物,纤维粉尘和臭氧作为肺的主要原因通过..活性氧物质的机制的代码诠释J ENVIRON RES公共卫生 10(9)癌变:3886至3907
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引用:Damulewicz, M. and Pyza, E. (2018). Determination of DNA Damage in the Retina Photoreceptors of Drosophila. Bio-protocol 8(3): e2708. DOI: 10.21769/BioProtoc.2708.
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