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Preparation of Precisely Oriented Cryosections of Undistorted Drosophila Wing Imaginal Discs for High Resolution Confocal Imaging
制备未畸变果蝇翅成虫盘精确定向冷冻切片以用于高分辨率共焦成像   

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Bio-protocol
Feb 2018

Abstract

The combination of immunofluorescence and laser scanning confocal microscopy (LSM) is essential to high-resolution detection of molecular distribution in biological specimens. A frequent limitation is the need to image deep inside a tissue or in a specific plane, which may be inaccessible due to tissue size or shape. Recreating high-resolution 3D images is not possible because the point-spread function of light reduces the resolution in the Z-axis about 3-fold, compared to XY, and light scattering obscures signal deep in the tissue. However, the XY plane of interest can be chosen if embedded samples are precisely oriented and sectioned prior to imaging (Figure 1). Here we describe the preparation of frozen tissue sections of the Drosophila wing imaginal disc, which allows us to obtain high-resolution images throughout the depth of this folded epithelium.


Figure 1. The epithelial structure and undistorted folding pattern are revealed in its entire depth in this frozen section of developing Drosophila wing. A-D. Transverse dorsoventral sections through the wing pouch. A. Cryosection reveals nuclei (A, green) and subcellular distribution of α-catenin (A’, A”, magenta) with signal throughout the depth of the epithelium. The basal surface is clearly detectable (arrows). A” is digitally enhanced image of A’. B. A Z-stack of images collected in a top-down view displayed as XZ orthogonal view reveals nuclei (B) but little discernable detail for α-catenin (B’, B”) and even the digitally enhanced image (B”) fails to reveal the basal epithelial surface (arrow). C. Transverse dorsoventral section displaying the Distal-less (Dll, green) gradient in the wing pouch and subcellular localization of DE-Cadherin (magenta) throughout the epithelium. D. View of the wing pouch. Dorsal is to the left; apical is up. Scale bars are 1 µm in A, B, 11 µm in C, 5 µm in D.

Keywords: Cryosection (冷冻超薄切片), Frozen sections (冰冻切片), Confocal microscopy (共聚焦显微镜技术), Wing imaginal disc (翅成虫盘), Drosophila (果蝇)

Background

Third instar imaginal discs are flat pocket-like involutions of the epidermis (Cohen, 1993; McClure and Schubiger, 2005). One layer of this pocket, the ‘disc proper’, is a pseudostratified columnar epithelium that is heavily folded at the onset of metamorphosis. It is continuous with the ‘overlaying’ squamous epithelium, the peripodial membrane. The focus of our work is to understand how the Wnt morphogen patterns the dome-shaped wing pouch region of the wing disc. As imaginal discs are flat overall, conventional imaging has them mounted for top-down or upside-down observation, whereby cover-slips compress and distort the folded structure. The use of spacers prevents distortions, but imaging of the entire wing pouch using Z-stacks has proved unsatisfactory or impossible; as outlined above, the reduced resolution in the Z-axis typically prevents high-resolution reconstruction of the epithelium in the apical/basal direction. Therefore, only the apical half of the epithelium of the wing pouch is detected at high resolution. This problem is exacerbated if weak signals are to be detected. Thus, uniform imaging requires a ‘side-view’ that can be obtained in sections. We modified a cryosection protocol (Culbertson et al., 2011; Sui et al., 2012) to obtain transverse sections of wing discs at defined angles. This methodology was critical to our analysis of signaling gradients in the wing pouch.

Materials and Reagents

  1. Pipette tips (USA Scientific, catalog numbers: 1111-1800 , 200 µl; 1111-2821 , 1,000 µl)
  2. Glass 9-well plate (PYREXTM Spot Plate, Fisher Scientific, catalog number: 13-748B )
  3. Razor blades (Single Edge Razor Blades, Stanley Black & Decker, catalog number: 28-510 )
  4. Conical tipped plastic embedding capsules (Electron Microscopy Sciences, BEEM®, catalog number: 69913-01 )
  5. Glass microscope slides (FisherfinestTM Premium Frosted, Fisher Scientific, catalog number: 12-544-2 )
  6. Microscope cover glass (coverslips), 22 x 50-1 (Fisher Scientific, Fisherbrand, catalog number: 12-545E )
  7. Plastic cling wrap
  8. Late third instar Drosophila melanogaster larvae
  9. Tissue Freezing Medium (TFMTM), clear (General Data, catalog number: TFM-C )
  10. Fluoromount-G® (SouthernBiotech, catalog number: 0100-01 )
  11. Clear Nail polish (i.e., Sally Hansen Hard As Nails)
  12. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271 )
  13. Potassium chloride (KCl) (Fisher Scientific, catalog number: P330 )
  14. Sodium bicarbonate (NaHCO3) (Fisher Scientific, catalog number: S233 )
  15. Calcium chloride dihydrate (CaCl2·2H2O) (Fisher Scientific, catalog number: C79 )
  16. Sodium phosphate dibasic anhydrous (Na2HPO4) (Fisher Scientific, catalog number: S374 )
  17. Potassium phosphate monobasic (KH2PO4) (Fisher Scientific, catalog number: P285 )
  18. Triton X-100 (Sigma-Aldrich, catalog number: X100 )
  19. Normal goat serum (Jackson ImmunoResearch, catalog number: 005-000-121 )
  20. Gelatin from porcine skin (gel strength 300 Type A, Sigma-Aldrich, catalog number: G2500 )
  21. D-Sucrose (Fisher Scientific, catalog number: BP220 )
  22. Sodium azide (Fisher Scientific, catalog number: S2271 )
  23. 16% paraformaldehyde (Ted Pella, catalog number: 18505 )
  24. Ice
  25. Dry ice
  26. Ringer’s solution (see Recipes)
  27. 4% formaldehyde fix (Solution A; see Recipes)
  28. 10x phosphate-buffered saline (PBS) (see Recipes)
  29. PBT (see Recipes)
  30. 5% normal goat serum (Solution B; see Recipes)
  31. 30% sucrose (Solution C; see Recipes)
  32. 10% gelatin (Solution D; see Recipes)

Equipment

  1. Dissection microscope (Leica Biosystems, model: Leica MZ6 )
  2. Fine forceps (Dumont Tweezers #5, World Precision Instruments, catalog number: 500085 )
  3. Pipettes 20 µl, 200 µl, 1,000 µl (Gilson, model: Pipetman P20, catalog number: F123600 ; Gilson, model: Pipetman P200, catalog number: F123601 ; Gilson, model: Pipetman P1000, catalog number: F123602 )
  4. Water bath, 50 °C (Fisher Scientific, model: IsotempTM 205 )
  5. Cryostat (Leica Biosystems, model: Leica CM1850 , with anti-roll plate assembly, Leica Biosystems, catalog number: 14041933981 )
    Note: The product “Leica CM1850” has been discontinued.
  6. Sample holders for cryostat (Specimen disc, 25 mm, Leica, catalog number: 14041619275 )
  7. Ultralow Temperature Freezer, -80 °C (Thermo Fisher Scientific, model: UXF60086A , catalog number: 315673H01)
  8. Fluorescence/confocal microscope (laser scanning microscope, ZEISS, model: LSM 780 )

Procedure

  1. Dissection and staining of wing imaginal discs
    Note: For a more detailed protocol, see Klein (2008).
    1. Under the dissection microscope: In chilled Ringer’s solution (see Recipes), dissect 3rd instar larvae, turning them inside out while leaving wing imaginal discs attached to the carcass. Remove as much unwanted tissue as possible (fat body, intestine, salivary glands)
    2. Fix in Solution A (made fresh; see Recipes) on ice for 20 min.
    3. Wash 3 x with PBT (see Recipes) each for 10 min.
    4. Block in Solution B (see Recipes) for 1 h at room temperature.
    5. Perform immunostaining following the standard protocol (Klein, 2008).
    6. Post-fix in Solution A for 1 h at room temperature.
    7. Wash 3 x with PBT each for 10 min.

  2. Embedding in gelatin
    1. In a glass 9-well plate, add 300 µl of 50 °C of Solution D (gelatin, see Recipes) to each well. Ensure that the surface of the solution is level and free of bubbles
    2. Place on ice for 30 min to allow the gel to solidify.
      Note: Try to prevent condensation from forming on glass 9-well plate, as falling droplets might dilute the gelatin solution and interfere with gelation.
    3. Under the dissection microscope: Gently remove the wing discs from carcasses in PBT.
    4. Using a pipette, transfer the wing discs into a well of Solution C (sucrose, see Recipes) and incubate for about 60 min at room temperature.
    5. Transfer a single wing disc by pipette (in about 20 µl of liquid) from the sucrose solution onto the solidified gelatin (Figure 2A).
      Note: The disc should be placed directly into the middle of the well.
    6. Remove as much excess liquid as possible, ensuring that the disc is lying flat on the gel (Figure 2B).
    7. Apply an additional 300 µl of 50 °C Solution D (gelatin) on top of the wing disc (Figure 2C).


      Figure 2. Schematic representation of the gelatin embedding process. A. Transfer a wing disc to the gelatin by pipette. B. Remove excess sucrose solution from around the disc. C. Add an addition 300 µl of gelatin on top of the disc.

    8. Place on ice and allow the gel to solidify about 30 min.
    9. Under a dissecting microscope, use a razor blade to cut the gelatin into a block around the sample.
      Note: This should be done with regard to the desired orientation of the cryosection. For example, if cross-sections are desired across the wing pouch dorso-ventrally, one face of the gel block should be perpendicular to the dorsoventral boundary (Figures 3A and 3C).


      Figure 3. Orientation of wing discs in cut gelatin cubes. Wing disc stained with DAB to visualize expression of Wingless & Patched, for illustration, in trimmed gelatin blocks. A. Top-down view of a wing disc in gelatin cube. The red line indicates the face of the cube which should be placed face-down into the embedding capsule to acquire a dorsoventrally aligned section. B. ‘edge-on’ view of the wing disc from the direction indicated by the arrow in A. C. An alternatively angled cube which would allow for sections aligned 45° to the dorsoventral boundary. The red line indicates face of the cube which should be placed downward.

    10. Prepare a plastic embedding capsule by cutting off the conical tip with a razor (Figures 4A-4B).
    11. Place the gel cube with the desired face down onto the lid of a plastic embedding capsule (Figures 3B and 4C).


      Figure 4. Preparation of plastic embedding capsule. A. Plastic embedding capsule (Step B8 above). B. Cut the conical tip off from the capsule using a razor blade (the arrow indicates movement of the blade). Discard the conical tip. C. Place the sample with the desired cutting face downward onto the lid of the capsule (red cube, indicated by arrow). D. Close the capsule and fill from the top with tissue freezing medium (arrow indicates where to add medium).

    12. Gently fill the capsule with tissue freezing medium, allowing large air bubbles to escape (Figure 4D).
    13. Place capsule immediately into dry ice or a -80 °C freezer.

  3. Cryosectioning
    1. Remove the frozen sample from the plastic embedding capsule. This is achieved by first removing the lid, then cutting away the remainder of the plastic capsule with a razor blade.
    2. Affix the sample to a cold (-80 °C) microtome sample holder using tissue freezing medium (Figures 5A-5B).
    3. With the cryostat chamber set to -24 °C, place the holder in the microtome (Figure 5C).
    4. Cut sections of 10-20 µm thickness. The anti-roll plate ensures a flat section comes off the knife (Video 1).
    5. Pick up tissue sections by gently touching a glass microscope slide to them. Create a row of sections (Video 1).
    6. Check under a fluorescence microscope for the presence of disc tissue in gelatin slices, and mark their location on the back of the slide.
    7. Add Fluoromount-G® atop the samples.
    8. Place a coverslip over the slide, seal with nail polish.


      Figure 5. Preparing samples for cryosectioning. A. An empty microtome sample holder; B. Sample affixed to holder using TFM; C. Holder with sample in the cryostat chamber.

      Video 1. Operation of the cryostat. The anti-roll plate, which is lowered at the beginning of the video, ensures the section remains flat. Note tissue sections will adhere to the microscope slide after gently coming in contact.

Data analysis

Laser scanning confocal microscopy can be used to examine tissue sections. For context on the morphology of tissue sections, see Note 1. For a comparison of cryosectioned wing discs and xz optical sectioning, see Figure 1.

Notes

  1. Several criteria can be used to judge whether the angle of the tissue section is correctly perpendicular or slightly oblique. The disc proper is a pseudostratified columnar epithelium. Nuclei are tightly packed in the nuclear layer, which occupies the apical half of the epithelium. Cells reach apical and basal surfaces via tube-like extensions. For transverse sections through the center of the wing pouch in the dorsoventral direction (Figure 7):
    1. The overall appearance of the wing pouch is symmetrical, with uniform height in the central part and relatively minor inward curvature near the ventral hinge region (Figure 7A).
    2. Sections through the nuclear layer have it appear about four nuclei deep and largely confined to the apical part of the epithelium (Figure 7A). Oblique sections through the dome-shaped epithelium may increase both thickness and position of the nuclear layer (Figures 7B and 7C).
    3. The epithelial height in the center of the wing pouch of the late third instar wing disc is approximately 35 µm (Figure 7). Oblique cuts are evident from a greater apparent height (Figure 7B).
    4. Cell outlines should be parallel (Figure 7A). Oblique cuts through cells reveal primarily the rounded outlines around nuclei, and the tube-like extensions that reach the apical and basal surface are not visible (Figures 7B and 7C).


      Figure 6. Tissue Section Examples. A. A near-perfect section through the wing pouch along the anteroposterior compartment boundary. The wing imaginal disc is a relatively flat sac, where the disc proper consists of a pseudostratified columnar epithelium; a simple cuboidal epithelium provides the transition to the simple squamous epithelium that makes up most of the (covering) peripodial membrane (PM). The wing pouch is dome-shaped and surrounded by folds of the future wing hinge (the ventral hinge region has a characteristic folding pattern in sections). The position of the dorsoventral compartment boundary is indicated (arrow, ‘D/V’); dorsal is to the left and ventral to the right. B. An oblique section through the wing pouch is evident from its uneven shape and nuclear layer, the epithelium is too high and of uneven height. C. Although largely in the dorsoventral direction, the section cuts slightly ‘diagonal’ across the disc. No clear parallel cell outlines are evident. The epithelium appears too high and of uneven height. Uneven depth and increased number of nuclei (> 4 nuclear profiles) identify an oblique cut. The sectioned wing pouch appears at an angle to the remainder of the disc, revealing a cut deviating from the dorsoventral axis. Scale bars are 15 µm in A, 9 µm in B, 12 µm in C.

  2. In cutting gelatin with embedded tissue (Figure 3), it is important that the gelatin remain cold and rigid during the cutting process to facilitate precise cuts and proper angling.
  3. For achieving correctly angled sections, the gelatin cutting and the cryosectioning steps are critical. During gelatin cutting, it is important to ensure the wing disc lies perfectly flat (Figure 3B). During cryosectioning, it is important to ensure the microtome blade is cutting evenly across the face of the sample.
  4. If excessive background is present in the fluorescence micrograph, the following may help: ensure that the gelatin is fresh; ensure that no dye is present in tissue freezing medium (clear TFM is recommended); ensure that the sample was post-fixed properly. To alleviate further background, tissue sections on the slide may be carefully rinsed with PBT before mounting with Fluoromount-G® and coverslip.
  5. Occasionally, holes or vacuoles may appear in the tissue. We believe these arise if mismatched ionic concentrations of solutions are used during the procedure.

Recipes

  1. Ringer’s solution
    For 1.0 L solution:
    6.5 g NaCl
    0.14 g KCl
    0.2 g NaHCO3
    0.16 g CaCl2
    0.01 g NaH2PO4
    Make up to 1,000 ml with deionized H2O and sterile filter
  2. 10x phosphate-buffered saline (PBS)
    For 1.0 L solution:
    80 g NaCl
    2 g KCl
    14.4 g Na2HPO4
    2.4 g KH2PO4
    Make up to 1,000 ml with deionized H2O and sterile filter
  3. PBT
    For 1.0 L solution:
    100 ml 10x PBS
    10 ml 10% Triton X-100
    Make up to 1,000 ml with deionized H2O and sterile filter
  4. Solution A: 4% formaldehyde in PBS
    For 1.0 ml solution:
    650 µl deionized H2O
    100 µl 10x PBS
    250 µl 16% paraformaldehyde
  5. Solution B: 5% goat serum in PBT
    For 1.0 ml solution:
    50 µl normal goat serum
    950 µl PBT
  6. Solution C: 30% sucrose solution
    For 50 ml solution
    15 g sucrose
    50 µl 10% sodium azide in H2O
    Make up to 50 ml with 1x PBS
  7. Solution D: 10% gelatin solution
    For 50 ml solution:
    5 g gelatin, gel strength 300, Type A
    15 g sucrose
    50 µl 10% sodium azide in H2O
    Make up to 50 ml with 1x PBS
    Place in 50 °C water bath to dissolve
    Store at 50 °C for short term or freeze for long term storage

Acknowledgments

We are grateful for discussions and improvements to this methodology to K. Hanson, J.M. Laumann, A. Snyder, M. Boardman, S. Kaech-Petrie, A. Nechiporuk and, in particular, M. Deza-Culbertson. This work was supported by grants of NIH (R01GM67029 & R01GM103876), NSF (IOS-1021573 & IOS-1353799), and the Medical Research Foundation (OR) to M.W. Authors have no conflicts of interest or competing interests.

References

  1. Cohen, S. M. (1993). Imaginal disc development. In: Bate, M. and Martinez Arias, A. (Eds.). The development of Drosophila melanogaster. Cold Spring Harbor Laboratory Press pp: 747-841.
  2. Culbertson, M. D., Lewis, Z. R. and Nechiporuk, A. V. (2011). Chondrogenic and gliogenic subpopulations of neural crest play distinct roles during the assembly of epibranchial ganglia. PLoS One 6(9): e24443.
  3. Klein, T. (2008). Immunolabeling of imaginal discs. In: Dahmann, C. (Ed.). Drosophila: Methods and Protocols. Methods Mol Biol 420: 253-263.
  4. McClure, K. D. and Schubiger G. (2005). Developmental analysis and squamous morphogenesis of the peripodial epithelium in Drosophila imaginal discs. Development 132(22): 5033-5042.
  5. Sui, L., Pflugfelder, G. O. and Shen, J. (2012). The Dorsocross T-box transcription factors promote tissue morphogenesis in the Drosophila wing imaginal disc. Development 139(15): 2773-2782.

简介

免疫荧光和激光扫描共聚焦显微镜(LSM)的组合是高分辨率检测生物样品中分子分布的关键。频繁的限制是需要在组织内或在特定的平面深处进行成像,这可能由于组织大小或形状而不可接近。因为与XY相比,光的点扩散函数将Z轴的分辨率降低了约3倍,并且光散射使组织中的深层信号模糊,所以不可能重新创建高分辨率3D图像。然而,如果嵌入的样品在成像之前被精确地定向和切片,则可以选择感兴趣的XY平面(图1)。在这里,我们描述的果蝇翅成像光盘的冰冻组织切片的准备,这使得我们能够获得高分辨率的图像,在整个这个折叠上皮的深度。

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图1.上皮结构和未畸变的折叠模式在发育果蝇翅膀的这个冰冻部分的整个深度中都被揭示出来。通过机翼囊横向背腹节。 A.冷冻切片显示贯穿上皮深度的信号的α-连环蛋白(A',A“,洋红色)的细胞核(A,绿色)和亚细胞分布。基底表面清晰可辨(箭头)。 A是“A的数字增强图像”。 B.在显示为XZ正交视图的自顶向下视图中收集的图像的Z-堆叠揭示了α-连环蛋白(B',B“)甚至数字增强图像(B”)的细胞核(B)但很少可辨别的细节。未能揭示基底上皮表面(箭头)。 C.横向背腹节在翼袋中显示无远端(Dll,绿色)梯度和DE-Cadherin(品红)在整个上皮中的亚细胞定位。 D.翼袋的视图。背在左边;顶端是起来的。比例尺在A,B中为1μm,C中为11μm,D中为5μm。


【背景】第三龄期的成像盘是表皮平坦的口袋状退化(Cohen,1993; McClure和Schubiger,2005)。这个口袋里的一层,“椎间盘本身”是一个假复层柱状上皮,在变态发作时大量折叠。与“覆盖”鳞状上皮,圆周膜连续。我们的工作重点是了解Wnt morphogen如何模仿翼片的圆顶形翼袋区域。由于成像盘总体上是平坦的,所以传统的成像将它们安装成自上而下或颠倒的观察,由此盖滑块压缩和扭曲折叠结构。使用隔离物可以防止变形,但是使用Z-堆叠成像整个机翼袋已被证明是不令人满意的或不可能的;如上所述,Z轴中降低的分辨率通常阻止顶部/基底方向上的上皮的高分辨率重建。因此,仅以高分辨率检测到翼袋上皮的顶端一半。如果要检测到微弱的信号,这个问题就会加剧。因此,均匀成像需要可以分段获得的“侧视”。我们修改了冰冻切片方案(Culbertson等人,2011; Sui等人,2012),以获得限定角度的翼片的横截面。这种方法对于我们对机翼袋中信号梯度的分析至关重要。

关键字:冷冻超薄切片, 冰冻切片, 共聚焦显微镜技术, 翅成虫盘, 果蝇

材料和试剂

  1. 移液管吸头(USA Scientific,目录号:1111-1800,200μl;1111-2821,1000μl)
  2. 玻璃9孔板(PYREX TM Spot Plate,Fisher Scientific,目录号:13-748B)
  3. 剃刀刀片(单刃剃刀刀片,斯坦利•布莱克和德克,目录号:28-510)
  4. 圆锥形塑料包埋胶囊(电子显微镜科学,BEEM ,目录号:69913-01)
  5. 玻璃显微镜载玻片(Fisherfinest TM Premium Frosted,Fisher Scientific,目录号:12-544-2)
  6. 显微镜盖玻片(盖玻片),22×50-1(Fisher Scientific,Fisherbrand,目录号:12-545E)
  7. 塑料保鲜膜
  8. 晚三分之一果蝇幼虫
  9. 组织冷冻培养基(TFM TM ),clear(通用数据,目录号:TFM-C)
  10. Fluoromount-G (SouthernBiotech,目录号:0100-01)
  11. 清除指甲油( ,Sally Hansen <硬指甲)
  12. 氯化钠(NaCl)(Fisher Scientific,目录号:S271)
  13. 氯化钾(KCl)(Fisher Scientific,目录号:P330)
  14. 碳酸氢钠(NaHCO 3)(Fisher Scientific,目录号:S233)
  15. 氯化钙二水合物(CaCl 2•2H 2 O)(Fisher Scientific,目录号:C79)
  16. 磷酸二氢钠无水(Na 2 HPO 4)(Fisher Scientific,目录号:S374)
  17. 磷酸二氢钾(KH 2 PO 4)(Fisher Scientific,目录号:P285)
  18. Triton X-100(Sigma-Aldrich,目录号:X100)
  19. 正常山羊血清(Jackson ImmunoResearch,目录号:005-000-121)
  20. 来自猪皮的明胶(凝胶强度300型A,Sigma-Aldrich,目录号:G2500)
  21. D-蔗糖(Fisher Scientific,目录号:BP220)
  22. 叠氮化钠(Fisher Scientific,目录号:S2271)
  23. 16%多聚甲醛(Ted Pella,目录号:18505)

  24. 干冰
  25. 林格的解决方案(见食谱)
  26. 4%的甲醛固定(解决方案A ;见食谱)
  27. 10倍磷酸盐缓冲盐水(PBS)(见食谱)
  28. PBT(见食谱)
  29. 5%正常山羊血清( ;见食谱)
  30. 30%蔗糖( C ;见食谱)
  31. 10%明胶(“D”溶液;见食谱)

设备

  1. 解剖显微镜(徕卡生物系统,型号:徕卡MZ6)
  2. 细钳(Dumont镊子#5,世界精密仪器,目录号:500085)
  3. 移液管20μl,200μl,1,000μl(Gilson,型号:Pipetman P20,目录号:F123600; Gilson,型号:Pipetman P200,目录号:F123601; Gilson,型号:Pipetman P1000,目录号:F123602)
  4. 水浴,50°C(Fisher Scientific,型号:Isotemp TM 205)
  5. 低温恒温器(Leica Biosystems,型号:Leica CM1850,带防卷板组件,Leica Biosystems,产品目录号:14041933981)
    注:产品“Leica CM1850”已停产。
  6. 低温恒温器样品架(样品盘,25 mm,Leica,目录号:14041619275)
  7. 超低温冰箱,-80°C(Thermo Fisher Scientific,型号:UXF60086A,目录号:315673H01)
  8. 荧光/共焦显微镜(激光扫描显微镜,蔡司,型号:LSM 780)

程序

  1. 翅膀成像盘的解剖和染色
    注:有关更详细的协议,请参阅Klein(2008)。
    1. 在解剖显微镜下:在冷冻林格溶液(参见食谱)中,解剖3龄幼虫,将它们向里翻出来,同时留下连接在尸体上的翅膀成像盘。去除尽可能多的有害组织(脂肪体,肠,唾液腺)

    2. 在冰上解决解决方案A (制作新鲜;参见食谱)20分钟。

    3. 每次用PBT清洗3次(见食谱)10分钟

    4. 在解决方案B (请参阅食谱)中于室温下封闭1小时
    5. 按照标准方案进行免疫染色(Klein,2008)。
    6. 在解决方案A 后在室温下修复1小时。
    7. 用PBT清洗3次,每次10分钟。

  2. 嵌入明胶
    1. 在玻璃9孔板中,向每个孔中加入300μl50℃的明胶溶液D(明胶,参见食谱)。确保溶液表面平整无气泡
    2. 放置在冰上30分钟以使凝胶凝固。
      注意:尽量避免在玻璃9孔板上产生冷凝,因为掉落的液滴可能会稀释明胶溶液并干扰凝胶。
    3. 在解剖显微镜下:轻轻地从PBT的尸体上取下翼片。
    4. 使用移液管,将翼片转移到 C(蔗糖,参见食谱)的一个孔中并在室温下孵育约60分钟。
    5. 通过移液管(约20μl液体)从蔗糖溶液转移到固化明胶上(图2A)。 注意:光盘应该直接放在井的中间。
    6. 去除尽可能多的液体,确保光盘平放在凝胶上(图2B)。
    7. 在翼片顶部再涂上300μl的50°C溶液D(明胶)(图2C)。


      图2.明胶包埋过程的示意图A.通过移液管将翼片转移到明胶上。 B.从圆盘周围除去多余的蔗糖溶液。 C.在圆盘上加入300μl明胶。

    8. 放在冰上,让凝胶凝固约30分钟。

    9. 在解剖显微镜下,使用刀片将明胶切成块状 注意:这应该根据冷冻切片的所需取向来完成。例如,如果需要穿过机翼袋背腹侧的横截面,凝胶块的一个面应该垂直于背腹边界(图3A和3C)。


      图3.切割明胶立方体中翅片的取向。用DAB染色的翼片可视化表达Wingless&amp;为了说明,在修剪过的明胶块中进行修补。 A.在明胶立方体中的翼片的俯视图。红线表示立方体的面部,其应该被面朝下地放置到包埋囊中以获得背腹对齐的部分。 B.从A.C.中的箭头所示的方向看翼片的“边缘”视图。一个交替倾斜的立方体,其将允许与背侧边界成45°的区段。红线表示应放置在下方的立方体的面。

    10. 准备一个塑料包埋胶囊,用剃刀切割圆锥形尖端(图4A-4B)。
    11. 将凝胶块放在塑料包埋胶囊的盖子上(图3B和4C)。


      图4.塑料包埋胶囊的制备:一种。塑料包埋胶囊(上述步骤B8)。 B.用剃刀刀片将锥形尖端从胶囊上切下(箭头指示刀片运动)。丢弃圆锥形尖端。 C.将所需的切割面向下放到胶囊盖上(红色方块,箭头所示)。 D.关闭胶囊并用组织冷冻介质从顶部填充(箭头表示添加介质的位置)。


    12. 使用组织冷冻介质轻轻地填充胶囊,使大量气泡逸出(图4D)
    13. 立即将胶囊放入干冰或-80°C冰箱中。

  3. 冷冻切片
    1. 从塑料包埋胶囊中取出冷冻的样品。这是通过首先取下盖子,然后用剃刀刀片切除塑料胶囊的其余部分来实现的。

    2. 使用组织冷冻培养基将样品固定在冷的(-80°C)切片机样品架上(图5A-5B)。
    3. 将低温恒温室设置为-24°C,将固定器放入切片机(图5C)。
    4. 切割10-20微米厚的切片。防卷板确保刀片(视频1)的平面部分脱落。
    5. 拿起组织切片,轻轻触摸玻璃显微镜载玻片。创建一排部分(视频1)。
    6. 在荧光显微镜下检查明胶切片中是否有椎间盘组织,并将其位置标记在切片背面。
    7. 在样品上添加Fluoromount-G ®。
    8. 将盖玻片放在幻灯片上,用指甲油密封。


      图5.准备用于冷冻切片的样品。 :一种。一个空的切片机样品架; B.使用TFM固定在固定器上的样品; C.在低温恒温箱中拿着样品。

      视频1

数据分析

激光扫描共聚焦显微镜可用于检查组织切片。有关组织切片的形态学背景,请参阅注释1.有关冷冻切片翼片与xz光学切片的比较,请参阅图1。

笔记

  1. 可以使用几个标准来判断组织切片的角度是正确的还是稍微倾斜的。椎间盘本身是假复层柱状上皮。细胞核紧密堆积在核层中,占据了上皮的顶端一半。细胞通过管状延伸到达顶端和基底表面。对于通过背囊中心的横向切片(图7):
    1. 翼袋的整体外观是对称的,在中心部分具有相同的高度,而在腹部铰链区域附近具有相对较小的向内曲率(图7A)。
    2. 穿过核层的切片出现约四个深的核,大部分局限于上皮的顶端部分(图7A)。通过圆顶形上皮的斜切片可以增加核层的厚度和位置(图7B和7C)。
    3. 晚期三龄翼瓣的翼袋中心的上皮高度约为35μm(图7)。
      从更高的表观高度可以明显看出斜切(图7B)
    4. 细胞轮廓应平行(图7A)。通过细胞的斜切口主要揭示细胞核周围的圆形轮廓,并且到达顶端和基底表面的管状延伸部分是不可见的(图7B和7C)。


      图6.组织切片示例A.通过沿着前后舱边界的翼袋的近乎完美的截面。翅膀的成像盘是一个相对平坦的囊,其中盘适当由假复层柱状上皮组成;一个简单的立方形上皮提供了转换到构成大部分(覆盖)周围膜(PM)的简单鳞状上皮。翼袋是圆顶形的并且被未来的翼铰链的折叠包围(腹侧铰链区域具有特征性的折叠图案)。指示背腹舱边界的位置(箭头,“D / V”);背面是左边,腹边是右边。 B.由于形状不规则,核层不均匀,通过翼袋的斜切面明显,上皮过高,高度不均匀。 C.尽管大部分在背腹方向上,但该部分在盘上略微“对角”切割。没有明确的平行单元轮廓是明显的。上皮显得过高而且高度不均匀。不均匀的深度和增加的核数(> 4个核剖面)确定了斜切。切片的翅膀袋与椎间盘的其余部分呈一定角度,显露出偏离背腹轴的切口。比例尺A为15μm,B为9μm,C为12μm。

  2. 在切割含有组织的明胶(图3)时,在切割过程中明胶保持冷却和刚性以促进精确切割和适当的倾斜是重要的。
  3. 为了实现正确的角度切片,明胶切割和冷冻切片步骤是至关重要的。在明胶切割过程中,重要的是要确保翼片完全平坦(图3B)。在冷冻切片过程中,确保切片刀片均匀地切割样品表面是很重要的。
  4. 如果荧光显微照片中存在过量的背景,可能有助于:确保明胶是新鲜的;确保组织冷冻介质中不存在染料(推荐使用清亮的TFM);确保样本正确后置。为了减轻进一步的背景,在使用Fluoromount-G 和盖玻片进行安装之前,可以使用PBT仔细冲洗载玻片上的组织切片。
  5. 偶尔,组织中可能会出现孔或空泡。我们相信,如果在该过程中使用不匹配的溶液离子浓度,则会出现这些问题。

食谱

  1. 林格的解决方案
    对于1.0升解决方案:
    6.5克NaCl
    0.14克KCl
    0.2克NaHCO 3•/ 2 0.16克CaCl 2 2 /
    0.01克NaH 2 PO 4 4 用去离子H 2 O和无菌过滤器补足至1000ml
  2. 10倍磷酸盐缓冲盐水(PBS)
    对于1.0升解决方案:
    80克NaCl
    2克KCl
    14.4克Na 2 HPO 4 4 2.4克KH 2 PO 4 4克/克 用去离子H 2 O和无菌过滤器补足至1000ml
  3. PBT
    对于1.0升解决方案:
    100毫升10倍PBS
    10毫升10%Triton X-100
    用去离子H 2 O和无菌过滤器补足至1000ml
  4. PBS中4%的甲醛
    对于1.0毫升的解决方案:
    650μl去离子H 2 O
    100μl10x PBS
    250μl16%多聚甲醛
  5. B液:5%山羊血清PBT
    对于1.0毫升的解决方案:
    50μl正常山羊血清
    950μlPBT
  6. Solution C :30%蔗糖溶液
    对于50毫升的解决方案
    15克蔗糖
    在H 2 O中50μl10%叠氮化钠 用1x PBS补足50 ml
  7. 溶液D :10%明胶溶液
    对于50毫升的解决方案:
    5克明胶,凝胶强度300,A型
    15克蔗糖
    在H 2 O中50μl10%叠氮化钠 用1x PBS补足50 ml
    置于50°C水浴中溶解
    短期储存在50°C或长期储存

致谢

我们感谢K. Hanson,J.M. Laumann,A. Snyder,M. Boardman,S. Kaech-Petrie,A. Nechiporuk和特别是M. Deza-Culbertson先生对这种方法的讨论和改进。这项工作得到了NIH(R01GM67029和R01GM103876),NSF(IOS-1021573和IOS-1353799)和医学研究基金会(OR)对M.W的资助。作者没有利益冲突或利益冲突。

参考

  1. Cohen,S.M。(1993)。 Imaginal光盘开发。在:贝特,米和马丁内斯阿里亚斯,答(编辑)。 果蝇的发展。冷泉港实验室出版社 pp:747-841。
  2. Culbertson,M. D.,Lewis,Z。R.和Nechiporuk,A. V.(2011)。 神经嵴的软骨形成和胶质细胞亚群在上臂神经节装配过程中起着不同的作用。 PLoS One 6(9):e24443。
  3. Klein,T。(2008)。 免疫标记成像盘在:Dahmann,C. (Ed。)。果蝇:方法和实验方法方法Mol Biol 420:253-263。
  4. McClure,K.D。和Schubiger G.(2005)。 果蝇果蝇的周围上皮的发育分析和鳞状形态发生。 开发 132(22):5033-5042。
  5. Sui,L.,Pflugfelder,G.O.and Shen,J。(2012)。 Dorsocross T-box转录因子促进果蝇翼的组织形态发生imaginal disc。 开发 139(15):2773-2782。
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引用:Petshow, S. . and Wehrli, M. (2018). Preparation of Precisely Oriented Cryosections of Undistorted Drosophila Wing Imaginal Discs for High Resolution Confocal Imaging. Bio-protocol 8(3): e2725. DOI: 10.21769/BioProtoc.2725.
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