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A Method to Injure, Dissect and Image Indirect Flight Muscle of Drosophila
果蝇间接飞行肌的损伤、解剖和成像方法   

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参见作者原研究论文

本实验方案简略版
eLIFE
Oct 2017

Abstract

Inducing an injury specifically to Drosophila flight muscles is a difficult task, owing to the small size of the muscles and the presence of the cuticle. The protocol described below provides an easy and reproducible method to induce injury in the Drosophila flight muscles.

Keywords: Drosophila flight muscles (果蝇飞行肌), Injury (损伤), Regeneration (再生), Insect satellite cells (昆虫卫星细胞)

Background

Muscles in vertebrates undergo regeneration, a process attributed to the Satellite cells, the resident stem cells. Our lab has recently shown that Drosophila flight muscles harbor stem cells similar to vertebrate satellite cells, namely insect satellite cells and show proliferation response to muscle injury (Chaturvedi et al., 2017). The ease of fly genetics and our method of inducing injury open up an opportunity to address relevant questions in the field of regenerative biology. We have standardized a protocol for injuring dorsal longitudinal muscles (DLMs) fibers with better precision, and this method can be used for investigating the repair mechanisms involved in muscles after the injury.

Materials and Reagents

  1. Very thin paint brush
  2. Minutien pins -Stainless Steel/0.1 mm Diameter (Minutien Pins) (Fine Science Tools, catalog number: 26002-10 )
  3. Standard fly food media in glass vial (e.g., BDSC Cornmeal Food)
  4. Standard Petri dish for immunostaining (e.g., FisherbrandTM Petri Dishes with Clear Lid, Thermo Fisher Scientific, catalog number: FB0875713 )
  5. Adult Drosophila melanogaster
  6. Cover slips (Thermo ScientificTM Gold SealTM Cover Slips) (Thermo Fisher Scientific, catalog number: 3306 )
  7. Frosted micro slides, Size: 75 mm long x 25 mm wide, Thickness: 1.35 mm (Blue Star, BLUE STAR (FROSTED MICRO SLIDES))
  8. Sharp razor blades (e.g., Gillette, 7 O'Clock Super Stainless BladesTM)
  9. Double sided adhesive tape (Mario Tapes, catalog number: SP 110 )
  10. Nail polish
  11. (Optional) Liquid nitrogen
  12. Ethanol (absolute for analysis EMSURE® ACS, ISO, Reag. Ph Eur) (Merck, catalog number: 1009831011 )
  13. Sodium chloride (NaCl) (Sodium Chloride, Fisher BioReagents) (Fisher Scientific, catalog number: BP358-1 )
  14. Potassium chloride (KCl) (Fisher Scientific, Potassium Chloride (Crystalline/USP/FCC), Fisher Chemical, catalog number: P330-500 )
  15. Sodium phosphate dibasic (Na2HPO4) (Fisher Scientific, Sodium Phosphate Dibasic Anhydrous (USP), Fisher Chemical, catalog number: S375-500 )
  16. Potassium phosphate monobasic (KH2PO4) (Potassium Phosphate Monobasic (Crystalline/Certified ACS)) (Fisher Scientific, Fisher Chemical, catalog number: P285-500 )
  17. Triton X-100 (Sigma-Aldrich, catalog number: X100-500ML )
  18. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: 05470-5G )
  19. Phalloidin (1:500 in 1x PBS) (Alexa Fluor 488® phalloidin) (Thermo Fisher Scientific, catalog number: A12379 )
  20. TOPRO-3-Iodide (1:1,000 in 1x PBS) (TO-PROTM-3 Iodide (642/661) - 1 mM Solution in DMSO) (Thermo Fisher Scientific, catalog number: T3605 )
  21. VECTASHIELD Antifade Mounting Medium (Vector laboratories, Vectashield®, catalog number: H-1000 )
  22. 16% Paraformaldehyde (PARAFORMALDEHYDE 16% Aqueous SOL. EM GRADE) (Electron Microscopy Sciences, catalog number: 15710 )
  23. (Optional) Anti-myosin
  24. (Optional) DAPI or Hoechst
  25. 70% (v/v) ethanol (see Recipes)
  26. 1x Phosphate buffered saline (PBS) (see Recipes)
  27. 16% Paraformaldehyde (PARAFORMALDEHYDE 16% Aqueous SOL. EM GRADE) (Electron Microscopy Sciences, catalog number: 15710 ) (see Recipes)
  28. Permeabilization solution (see Recipes)
  29. Blocking solution (see Recipes)

Equipment

  1. Stereo microscope (Olympus, model: SZX12 ) equipped with an imaging system (light source: Olympus KL1500 LCD, camera: QIClickTM CCD Camera, model: 01-QICLICK-R-F-CLR-12 , image acquisition software: Micromanager 1.4)
  2. CO2 pad (e.g., FlyStuff flypad, Genesee Scientific, catalog number: 59-114 )
  3. A Moria Nickel Plated Pin Holder (Moria Nickel Plated Pin Holder) (Fine Science Tools, catalog number: 26016-12 )
  4. Forceps (Dumont #5 Forceps) (e.g., Fine Science Tool, catalog number: 11251-10 )
  5. Scissor (Vannas Spring Scissors–2 mm Cutting Edge) (e.g., Fine Science Tools, catalog number: 15000-03 )
  6. Laser Scanning Confocal Microscope Olympus FV1000 (FluoView® FV3000 Confocal Laser Scanning Microscope) (e.g., Olympus, model: FV3000 )
  7. pH meter

Software

  1. Image acquisition software:
    1. Olympus Fluoview 1000 for Laser Scanning Confocal Microscope
    2. Micromanager 1.4 for recording the video
  2. Image processing software:
    1. Fiji
    2. GNU Image Manipulation Program (GIMP)

Procedure

  1. Fly muscle injury
    1. Take one Minutien pin with a pin holder as shown in Figure 1B.
    2. Clean the pin with 70% v/v ethanol and then with distilled water and dry to avoid contamination. Repeat this procedure every time before pricking the fly with the pin.
    3. Take 1-2 day old adult Drosophila and anesthetize them with CO2 on a CO2 pad (Figure 1A). Avoid taking more than 10-15 flies on a pad.
      Note: Avoid overexposure of CO2 to the flies. Use of cold anesthesia is not suitable for this process as it might have unwanted cold injury effects. This can significantly alter the results.
    4. Place the fly laterally as shown in Figure 1A under a stereo microscope and hold the thorax-abdomen junction gently with the light thin paint brush in one hand.
      Note: Avoid use of excessive force while orienting and holding the flies for inducing injury.
    5. While holding the fly, prick the thorax with the pin in PS (presutural) region as circled in Figure 1A. See the Video 1 as a reference.

      Video 1. Injuring DLMs by a single pinprick at PS (presutural) region. For demonstration purpose, the fly was immobilized using a little drop of nail polish on a glass slide. During actual experiments use of any adhesive is unnecessary. As shown in the video, we make a single prick at PS (presutural) location highlighted with a red arrow (Also shown in Figure 1A). To minimize damage to surrounding tissue, it is crucial to be quick and strictly to avoid moving the pin once it pierces fly thorax. This video was acquired using Stereo microscope coupled with a camera (see Equipment section for technical details). For image acquisition, an open source software micromanager (μManager) has been used. For further reference see Edelstein et al., 2014. Images and videos were processed using Fiji.

    6. Pricking by the pin should be at an angle of 45° to the A-P axis (anterior-posterior axis is the line running from head to abdomen that divides the animal with bilateral symmetry) so that only DLMs get injured (Figure 1C).
      Alternative step: Before pricking the thorax, the pin can be cooled by liquid nitrogen by dipping for a brief moment.
    7. The thorax should be injured with a pin by inserting ~0.5 mm so that only one of the hemithorax get injured (Figure 1D). This will ensure stem cell activation preferably in that hemithorax (Figure 1E) (Chaturvedi et al., 2017).
      Note: After the injury, there will be melanization which can be easily identified as a black spot.
    8. Transfer flies into food vial for recovery and process them for immunostaining after 5-6 h of recovery at least (Figures 1D and 1E).


      Figure 1. An image guide for inducing injury at Dorsal Longitudinal Muscle. A. Canton-S fly showing the site of injury marked by a white circle; B. A pin with holder for inducing injury, Scale bar = 1 cm. C. Schematic of longitudinal section of fly thorax. The arrow depicts the direction of pin inducing injury to DLM (Dorsal longitudinal muscles, depicted by six purple rectangles). Arrowhead shows the point of contact between a pin and muscle fiber. Green and Blue boxes represent other flight muscles in thorax that are left mostly uninjured. D. Thoracic flight muscles (DLM) showing the site of an injury indicated by a dotted circle (white). Whole mount of flight muscles labeled by phalloidin (green) with all nuclei labeled by TOPRO-3 (blue). Scale bar = 50 µm. E. Simplified scheme depicting unfused muscle stem cells associated with flight muscles.

  2. Fly DLM dissection
    1. Take one adult injured Drosophila and anesthetize.
      Note: A semi-alternative method has been given as reference (Weitkunat and Schnorrer, 2014).
    2. Put the anesthetized fly in a petri dish containing 1x PBS (pH 7.5) and use sufficient amount of 1x PBS to keep the animal submerged.
    3. Hold the fly abdomen with forceps and cut the head, legs, wings, and abdomen with fine scissors under a stereo microscope. Keep only the thoracic part of the fly for rest of the procedure.
      Note: To avoid accidental poking into muscles, operate thoracic tissue henceforth using leg stumps or halteres.

  3. Immunohistochemistry and confocal microscopy
    1. Submerge the whole thorax in 4% PFA in 1x PBS taken in a Petri dish for chemical fixation for 20 min (see Recipes).
    2. Take a glass slide and stick double sided tape on the slide.
    3. Place the thorax on the double-sided tape slide and orient the fly thorax in such a way that the ventral side is up. Then stick it on the tape.
    4. Make a sagittal cut with a fine razor blade/scissor by following the ventral midline and submerge the hemi-thoraces in 1x PBS solution by holding wings or legs using forceps.
      Note: Try to use finely sharpened new blades for better sectioning.
    5. Wash the hemi-thoraces by submerging in permeabilization solution.
    6. Essentially perform the immunostaining and microscopy as mentioned in Fernandes et al., 1991 and Chaturvedi et al., 2017.
    7. For staining F-actin, Phalloidin has been used at 1:500 dilution in 1x PBS.
    8. For staining nuclei, TOPRO-3-Iodide has been used at 1:1,000 dilution in 1x PBS.

  4. Limitations of the method and expertise required
    1. If a pin fails to poke through cuticle – The pin has lost its sharpness and replace it with a new pin. A new pin should be used for every set of 20-30 flies for consistent results.
    2. Diameter of injured area differs between flies – Try a new pin.
    3. Death of Drosophila post injury – Avoid extensive injury to trachea on the ventral side of the fly.
    4. Infection/yeast growth in the injured area – Sterilize the pin and holder using 70% ethanol.
    5. Staining is too faint – Remove excess of tissue such as abdomen, head, and legs.
    6. Over-fixation of samples can result in absent or low staining. To obtain better results, samples should be fixed only for 20 min using 4% paraformaldehyde (always freshly prepared from 16% stock using 1x PBS).
    7. Muscle shows the absence of F-actin staining – Muscle injury can lead to areas of low or no phalloidin staining. Alternatively, anti-myosin staining can be used to observe the overall muscle structure.
    8. The preferred way to stain all the nuclei is to use DAPI/Hoechst along with secondary antibody rather than mounting media with DAPI in it.

Data analysis

Image analysis and data processing were essentially performed as mentioned in Chaturvedi et al., 2017.

Recipes

  1. 70% (v/v) ethanol in ddH2O
  2. 1x Phosphate buffered saline (PBS) (Cold Spring Harbor Protocols)
    NaCl 137 mM
    KCl 2.7 mM
    Na2HPO4 10 mM
    KH2PO4 1.8 mM
    Adjust pH to 7.5
  3. 4% paraformaldehyde for chemical fixation
    Make 4% paraformaldehyde from 16% paraformaldehyde in 1x PBS
  4. Permeabilization solution
    PBS containing 0.3% Triton X-100
  5. Blocking solution
    PBS containing 0.3% Triton X-100 and 0.1% of bovine serum albumin (BSA)

Acknowledgments

This protocol was adapted from the article, Identification and functional characterization of muscle satellite cells in Drosophila by Chaturvedi et al., 2017. We thank National Centre for Biological Sciences, Tata Institute of Fundamental Research and the J C Bose Fellowship of the Government of India for funding. We acknowledge Central Imaging & Flow Cytometry Facility for using the confocal microscope and NCBS Fly facility. The authors are also thankful to Avishek Ghosh and Rajan Surendra Thakur from Prof. Raghu Padinjat’s laboratory for helping us to use the stereo microscope with a camera attachment. We have no conflict of interest to declare.

References

  1. Chaturvedi, D., Reichert, H., Gunage, R. D. and VijayRaghavan, K. (2017). Identification and functional characterization of muscle satellite cells in Drosophila. Elife 6.
  2. Fernandes, J., Bate, M. and Vijayraghavan, K. (1991). Development of the indirect flight muscles of Drosophila. Development 113(1): 67-77.
  3. Edelstein, A. D., Tsuchida, M. A., Amodaj, N., Pinkard, H., Vale, R. D. and Stuurman, N. (2014). Advanced methods of microscope control using μManager software. J Biol Methods 1(2).
  4. Weitkunat, M. and Schnorrer, F. (2014). A guide to study Drosophila muscle biology. Methods 68(1): 2-14.

简介

由于肌肉尺寸小和角质层的存在,特别针对果蝇飞行肌肉诱导损伤是一项艰巨的任务。 下面描述的方案提供了一种简单且可重复的方法来诱导果蝇飞行肌肉中的损伤。

【背景】脊椎动物的肌肉进行再生,这是一种归因于卫星细胞(即常驻干细胞)的过程。 我们的实验室最近表明,果蝇的肌肉具有与脊椎动物卫星细胞相似的干细胞,即昆虫卫星细胞,并显示对肌肉损伤的增殖反应(Chaturvedi等人,2017年))。 飞蝇遗传学的易用性和我们的诱导伤害的方法为解决再生生物学领域的相关问题提供了机会。 我们已经标准化了一种以更好的精确度损伤背侧纵肌(DLMs)纤维的方案,并且该方法可以用于研究损伤后肌肉中涉及的修复机制。

关键字:果蝇飞行肌, 损伤, 再生, 昆虫卫星细胞

材料和试剂

  1. 非常薄的油漆刷
  2. Minutien别针 - 不锈钢/ 0.1毫米直径(Minutien Pins)(精细科学工具,目录号:26002-10)
  3. 在玻璃瓶中的标准飞行食物媒介(,例如,BDSC玉米面食物)
  4. 用于免疫染色的标准培养皿(例如,Fisherbrand TM Petri Dishes with Clear Lid,Thermo Fisher Scientific,目录号:FB0875713)
  5. 成人果蝇
  6. 盖玻片(Thermo Scientific TM TM Gold Seal TM Cover Slips)(Thermo Fisher Scientific,目录号:3306)
  7. 磨砂微型幻灯片,尺寸:75毫米长×25毫米宽,厚度:1.35毫米(蓝星,蓝星(FROSTED MICRO SLIDES))
  8. 锋利的剃须刀刀片(,例如,吉列,7个超级不锈钢刀片TM )
  9. 双面胶带
    https://www.scotchbrand.com/3M/en_US/scotch-brand/products/目录/〜/?N = 4335 + 3293674058 + 3294529207 + 3294857497& rt = rud
  10. 指甲油
  11. (可选)液氮
  12. 乙醇(绝对用于分析EMSURE ACS,ISO,Reag。Ph Eur)(Merck,目录号:1009831011)
  13. 氯化钠(NaCl)(氯化钠,Fisher BioReagents)(Fisher Scientific,目录号:BP358-1)
  14. 氯化钾(KCl)(Fisher Scientific,氯化钾(结晶/ USP / FCC),Fisher Chemical,目录号:P330-500)
  15. 磷酸二氢钠(Na 2 HPO 4)(Fisher Scientific,无水磷酸钠(USP),Fisher Chemical,目录号:S375-500)
  16. 磷酸二氢钾(KH 2 PO 4)(磷酸二氢钾(结晶/认证的ACS))(Fisher Scientific,Fisher Chemical,目录号:P285-500) />
  17. Triton X-100(Sigma-Aldrich,目录号:X100-500ML)
  18. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:05470-5G)
  19. 鬼笔环肽(在1x PBS中1:500)(Alexa Fluor 488鬼笔环肽)(Thermo Fisher Scientific,目录号:A12379)
  20. TOPRO-3-碘化物(在1×PBS中1:1,000)(TO-PRO TM -3碘化物(642/661) - 在DMSO中的1mM溶液)(Thermo Fisher Scientific,目录号:T3605)

  21. VECTASHIELD Antifade安装介质(Vector laboratories,Vectashield ®,目录号:H-1000)
  22. 16%多聚甲醛(PARAFORMALDEHYDE 16%水溶液EM级)(Electron Microscopy Sciences,目录号:15710)
  23. (可选)抗肌球蛋白
  24. (可选)DAPI或Hoechst
  25. 70%(v / v)乙醇(见食谱)
  26. 1x磷酸盐缓冲盐水(PBS)(见食谱)
  27. 16%多聚甲醛(PARAFORMALDEHYDE 16%水溶液EM级)(Electron Microscopy Sciences,目录号:15710)(见食谱)
  28. 透化溶液(见食谱)
  29. 阻止解决方案(请参阅食谱)

设备

  1. 装备有成像系统(光源:Olympus KL1500 LCD,相机:QIClick TM CCD相机,型号:01-QICLICK-RF-CLR-12)的立体显微镜(Olympus,型号:SZX12)软件:Micromanager 1.4)
  2. (例如,,FlyStuff flypad,Genesee Scientific,目录号:59-114)
  3. Moria镀镍针座(Moria镀镍针座)(精细科学工具,目录编号:26016-12)
  4. 镊子(Dumont#5镊子)(例如,Fine Science Tool,目录号:11251-10)
  5. 剪刀(Vannas Spring Scissors-2 mm Cutting Edge)(例如,Fine Science Tools,产品目录号:15000-03)
  6. 激光扫描共聚焦显微镜奥林巴斯FV1000(FluoView FV3000共聚焦激光扫描显微镜)(例如,奥林巴斯,型号:FV3000)
  7. pH计

软件

  1. 图像采集软件:
    1. 奥林巴斯Fluoview 1000激光扫描共聚焦显微镜
    2. Micromanager 1.4用于录制视频
  2. 图像处理软件:
    1. 斐济
    2. GNU图像操作程序(GIMP)

程序

  1. 飞肌肉受伤

    1. 如图1B所示,将一个Minutien针与一个针座连接起来
    2. 用70%v / v乙醇清洁针头,然后用蒸馏水清洗并干燥以避免污染。每次在用针刺动苍蝇前重复此步骤。
    3. 取1-2天龄的成年果蝇并在CO 2垫上用CO 2将它们麻醉(图1A)。避免在垫上摄取超过10-15只苍蝇。
      注意:避免苍蝇过度曝光CO 2 。冷麻醉的使用不适合这个过程,因为它可能有不必要的冷伤害效应。这可以显着改变结果。
    4. 如图1A所示,在体视显微镜下横向放置苍蝇,一手拿着轻薄的油漆刷轻轻握住胸腹交界处。
      注意:避免使用过度的力量,同时定位和抓住苍蝇以诱发受伤。
    5. 在保持苍蝇的同时,用图1A中圈出的PS(前尿道)区域的针戳刺胸腔。请参阅视频1作为参考。

      视频1
    6. 刺针应该与A-P轴成45°角(前后轴是从头部到腹部的线,将动物分为两侧对称),这样只有DLM受伤(图1C)。
      替代步骤:在刺破胸部之前,可以通过浸渍短暂的时间用液氮冷却针。
    7. 插入〜0.5 mm的胸部应该用销钉将其损伤,使得只有一个胸腔受伤(图1D)。这将确保干细胞活化,优选在该半胸内(图1E)(Chaturvedi等人,2017)。
      注:受伤后,会出现黑化现象,很容易识别为黑点。
    8. 将苍蝇转移到食品瓶中进行回收,至少在恢复5-6小时后处理它们进行免疫染色(图1D和1E)。


      图1.在背侧纵肌中诱导损伤的图像指导A.广州-S飞行显示白色圆圈标记的损伤部位; B.带支架的销钉引起损伤,比例尺= 1厘米。 C.飞行胸部纵剖面示意图。箭头描述了引起DLM损伤的针的方向(背侧纵肌,由六个紫色矩形描绘)。箭头显示针和肌肉纤维之间的接触点。绿色和蓝色框代表胸部的其他飞行肌肉,大部分未受伤。 D.胸部飞行肌肉(DLM)显示由虚线圆圈(白色)指示的损伤部位。由鬼笔环肽标记的整个飞行肌肉(绿色)与所有由TOPRO-3(蓝色)标记的核素。比例尺= 50μm。 E.简化的方案描述与飞行肌肉相关的未融合的肌肉干细胞。

  2. 飞DLM解剖:
    1. 让一名成年受伤的果蝇 并麻醉。
      注:一个半替代方法已被作为参考(Weitkunat和Schnorrer,2014)。
    2. 将麻醉后的苍蝇置于含有1x PBS(pH 7.5)的培养皿中,并使用足够量的1x PBS以保持动物不被淹没。
    3. 用镊子夹住苍蝇腹部,并在体视显微镜下用精细剪刀剪下头部,腿部,翅膀和腹部。
      只保留苍蝇的胸部 注意:为避免意外戳入肌肉,此后应使用腿部残肢或臀部操作胸部组织。

  3. 免疫组化和共聚焦显微镜:
    1. 将整个胸腔浸入1%PBS中的4%PFA中,置于培养皿中化学固定20分钟(见食谱)。
    2. 拿一张玻璃幻灯片,并在幻灯片上贴上双面胶带。
    3. 将胸部放在双面胶带上,并将胸部的方向定位,使腹侧向上。然后将其粘贴在磁带上。
    4. 按照腹侧中线,用精细剃刀刀片/剪刀进行矢状切割,并使用镊子握住翅膀或腿部,将1半PBS溶液中的半胸部浸入水中。
      注意:请尝试使用精细锐化的新刀片进行更好的切片。

    5. 通过浸透透化溶液清洗半胸部
    6. 基本上执行如1991年Fernandes等人和Chaturvedi 等人中所述的免疫染色和显微术。
    7. 为了染色F-肌动蛋白,已经在1x PBS中以1:500稀释度使用鬼笔环肽。
    8. 为了染色细胞核,TOPRO-3-碘化物在1x PBS中以1:1,000稀释使用。

  4. 所需方法和专业知识的局限性:
    1. 如果一个针不能通过角质层戳 - 该针已经失去了清晰度,并用一个新针来代替。
      每一套20-30只苍蝇都应该使用一个新的针以获得一致的结果。

    2. 。不同的苍蝇受伤地区的直径不同 - 尝试新的针。
    3. 果蝇死亡伤害后 - 避免苍蝇腹侧广泛受伤。
    4. 感染/酵母在受伤部位的生长 - 使用70%乙醇消毒针和支架。
    5. 染色太微弱 - 去除多余的组织,如腹部,头部和腿部。
    6. 样本过度固定可能导致无染色或低染色。为了获得更好的结果,应使用4%多聚甲醛(总是使用1x PBS从16%储液新鲜制备)将样品固定20分钟。
    7. 肌肉显示不存在F-肌动蛋白染色 - 肌肉损伤可导致低或无鬼菌素染色的区域。另外,抗肌球蛋白染色可以用来观察整体肌肉结构。
    8. 染色所有细胞核的首选方法是将DAPI / Hoechst与二抗一起使用,而不是使用含有DAPI的培养基。

数据分析

图像分析和数据处理基本上按照Chaturvedi等人在2017年提到的方法进行。

食谱

  1. 在ddH 2 O中70%(v / v)乙醇
  2. 1x磷酸盐缓冲液(PBS)(冷泉港协议) /> NaCl 137mM
    KCl 2.7 mM
    Na 2 HPO 4 4 10mM
    KH 2 PO 4 4 1.8mM 调整pH值至7.5
  3. 4%多聚甲醛用于化学固定

    在1x PBS中由16%多聚甲醛制成4%多聚甲醛
  4. 透化解决方案
    含有0.3%Triton X-100的PBS
  5. 阻止解决方案
    含有0.3%Triton X-100和0.1%牛血清白蛋白(BSA)的PBS

致谢

该协议摘自2017年由Chaturvedi等人撰写的文章“果蝇中肌肉卫星细胞的鉴定和功能特性鉴定”。我们感谢国家生物科学中心,塔塔研究所基础研究和JC Bose奖学金获得印度政府资助。我们承认Central Imaging& Flow Cytometry Facility使用共焦显微镜和NCBS Fly设备。作者还感谢来自Raghu Padinjat教授实验室的Avishek Ghosh和Rajan Surendra Thakur,他帮助我们将立体显微镜与相机附件一起使用。我们没有利益冲突要申报。

参考

  1. Chaturvedi,D.,Reichert,H.,Gunage,R.D。和VijayRaghavan,K.(2017)。 果蝇肌肉卫星细胞的鉴定和功能鉴定。 > Elife 6.
  2. Fernandes,J.,Bate,M.和Vijayraghavan,K.(1991)。 发展果蝇的间接飞行肌肉 发展 113(1):67-77。
  3. Edelstein,A.D.,Tsuchida,M.A.,Amodaj,N.,Pinkard,H.,Vale,R.D。和Stuurman,N.(2014)。 使用μManager软件进行显微镜控制的先进方法 J Biol方法 1(2)。
  4. Weitkunat,M.和Schnorrer,F。(2014)。 学习果蝇肌肉生物学的指南 方法 68(1):2-14。
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Copyright Chakraborty et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Chakraborty, K., VijayRaghavan, K. and Gunage, R. (2018). A Method to Injure, Dissect and Image Indirect Flight Muscle of Drosophila. Bio-protocol 8(10): e2860. DOI: 10.21769/BioProtoc.2860.
  2. Chaturvedi, D., Reichert, H., Gunage, R. D. and VijayRaghavan, K. (2017). Identification and functional characterization of muscle satellite cells in Drosophila. Elife 6.
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