参见作者原研究论文

本实验方案简略版
Oct 2017

本文章节


 

Preserve Cultured Cell Cytonemes through a Modified Electron Microscopy Fixation
通过一种改良的电子显微技术固定方法保存培养的细胞导管   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Immunocytochemistry of cultured cells is a common and effective technique for determining compositions and localizations of proteins within cellular structures. However, traditional cultured cell fixation and staining protocols are not effective in preserving cultured cell cytonemes, long specialized filopodia that are dedicated to morphogen transport. As a result, limited mechanistic interrogation has been performed to assess their regulation. We developed a fixation protocol for cultured cells that preserves cytonemes, which allows for immunofluorescent analysis of endogenous and over-expressed proteins localizing to the delicate cellular structures.

Keywords: MEM-fix (MEM固定), Cytonemes (细胞导管), Filopodia (丝状伪足), Microscopy (显微镜观察), Immunofluorescence (免疫荧光), Morphogen (形态发生素)

Background

Cytonemes are classified as thin (~200 nm diameter) actin based filopodia, over 2 μm in length, which can transport morphogens (Ramírez-Weber and Kornberg, 1999). These signaling structures were first classified and described in detail in the developing Drosophila wing imaginal disc, and have subsequently been observed in mouse, chick and zebrafish model organisms (Ramírez-Weber and Kornberg, 1999; Sanders et al., 2013; Stanganello et al., 2015). In most of these cases, cytoneme detection was only possible with live imaging of over-expressed, fluorescently labeled proteins. Examination of cytonemes of cultured cells has been limited due to traditional fixation protocols failing to preserve these fragile filaments. These complications have been limiting factors in determining the cellular mechanisms driving cytoneme formation and function during development and tissue homeostasis, and determining whether these processes are corrupted in disease.

In order to overcome these limitations, we developed a modified electron microscopy fixative (MEM-fix)-based protocol that preserves cytonemes of cultured cells. Use of MEM-fix allows for the detection of endogenous and over-expressed proteins of interest in the filopodial structures via traditional immunofluorescent protocols (Bodeen et al., 2017). MEM-fix is generated by the addition of glutaraldehyde to a final working concentration of 0.5% to a standard 4% paraformaldehyde fixative solution. Glutaraldehyde, which is commonly used to fix cells for electron microscopy-based studies, was included because of its ability to effectively preserve subcellular structures. Although glutaraldehyde is not an optimal fixative for immunofluorescence microscopy due to its propensity to auto-fluoresce, we determined that addition of 26.4 mM sodium borohydride to the permeabilization buffer was sufficient to mitigate this undesirable side effect (Tagliaferro et al., 1997; Bacallao et al., 2006). Unfortunately, due to glutaraldehyde limiting antibody penetration into cells, MEM-fix is not conducive to staining of cytoplasmic or nuclear proteins, so should be limited to an examination of integral membrane or juxta-membrane proteins.

We recently used this technique to examine Hedgehog (Hh) morphogen transport through cytonemes of cultured Drosophila cells and mouse fibroblasts (Bodeen et al., 2017). Here, we provide an optimized protocol for imaging of cytonemes in NIH3T3 cells, and provide examples of its adaptability to additional cultured mammalian cell lines (Figure 1). MEM-fix protocol modifications to standard cell fixation methods allow for reproducible detection of cytonemes and immunofluorescence-based staining of trans-membrane and membrane-adjacent proteins in cultured cells. MEM-fix also preserves signal of fluorescently-labeled proteins, so is not solely dependent on immuno-detection based methods.


Figure 1. MEM-fix increases preservation of cytonemes in cultured cells compared to paraformaldehyde fixation. A and B. HEK293T cells transfected with Shh. A. Cells fixed with paraformaldehyde show some actin-based protrusions, but do not show Shh (green) positive filaments. B. Cells fixed with MEM-fix contain many cytoneme projections, marked by the presence of F-actin (red) and Shh. Scale bars = 25 µm.

Materials and Reagents

  1. SHARP® Precision Barrier Tips, For P-1000 and Eppendorf 1,000, 1,250 µl (Denville Scientific, catalog number: P1126 )
  2. SHARP® Precision Barrier Tips, For P-200, 200 µl (Denville Scientific, catalog number: P1122 )
  3. SHARP® Precision Barrier Tips, For P-20, 20µl (Denville Scientific, catalog number: P1121 )
  4. SHARP® Precision Barrier Tips, Extra Long for P-2 and P-10, 10 µl (Denville Scientific, catalog number: P1096-FR )
  5. Gold SealTM Rite-OnTM Micro Slides (Thermo Fisher Scientific, catalog number: 3050-002 )
  6. 12 mm Microscope Cover Glass-1.5 (Fisher Scientific, catalog number: 12-545-81 )
  7. TPP® centrifuge tubes, volume 50 ml, polypropylene (TPP Techno Plastic Products, catalog number: 91050 )
  8. TPP® centrifuge tubes, volume 15 ml, polypropylene (TPP Techno Plastic Products, catalog number: 91015 )
  9. StericupTM Sterile Vacuum Filter Units 500 ml (Merck, catalog number: SCGPU05RE )
  10. 24-well plate (Corning, Falcon®, catalog number: 353226
  11. 6-well plate NunclonTM Delta Surface (Thermo Fisher Scientific, catalog number: 140675 )
  12. Professional Kimtech ScienceTM KimwipesTM (KCWW, Kimberly-Clark, catalog number: 34155 )
  13. Premium Microcentrifuge Tubes: 1.5 ml (Fisher Scientific, catalog number: 05-408-129 )
  14. NIH3T3 (ATCC, catalog number: CRL-1658 )
  15. Ultrapure water
  16. DMEM (1x) 4.5 g/L D-glucose, [-] L-Glutamine, [-] HEPES, [-] Sodium Pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 11960044 )
  17. Opti-MEMTM Reduced Serum Medium (Thermo Fisher Scientific, catalog number: 31985070 )
  18. Pen/Strep, 100x (Merck, catalog number: TMS-AB2-C )
  19. HyCloneTM Cosmic CalfTM Serum (BCS) (GE Healthcare, catalog number: SH30087.03 )
  20. MEM NEAA (100x) MEM Non-Essential Amino Acids (Thermo Fisher Scientific, GibcoTM, catalog number: 11140050 )
  21. L-Glutamine (100x) (100 ml) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 25030081 )
  22. Sodium Pyruvate (100 mM) 100x (Thermo Fisher Scientific, GibcoTM, catalog number: 11360070 )
  23. Lipofectamine® 3000 transfection kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: L3000-015 )
  24. 70% (volume) ethanol diluted in water
  25. 0.05% Trypsin 0.53 nM EDTA, 1x [-] Sodium Bicarbonate (Corning, catalog number: 25-052-Cl )
  26. DPBS, 1x (Dulbecco's Phosphate-Buffered Saline) (Corning, CellgroTM, catalog number: 21-031-CM )
  27. Sodium phosphate dibasic (Sigma-Aldrich, catalog number: S0876 )
  28. Sodium phosphate monobasic (Sigma-Aldrich, catalog number: S5011 )
  29. Sodium borohydride (Sigma-Aldrich, catalog number: 213462-25G )
  30. Formaldehyde, 16%, methanol free, Ultra Pure EM Grade (Polysciences, catalog number: 18814-10 )
  31. Glutaraldehyde, 8% Aqueous Solution, EM Grade (Electron Microscopy Sciences, catalog number: 16019 )
  32. Normal Goat Serum (10 ml) (Jackson ImmunoResearch, catalog number: 005-000-121 )
  33. Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
  34. Tween 20 (Acros Organics, catalog number: AC233360010
  35. Sonic hedgehog (Shh) antibody (H-160), rabbit polyclonal (Santa Cruz Biotechnology, catalog number: sc-9024 )
  36. Alexa FluorTM 633 Phalloidin (Thermo Fisher Scientific, catalog number: A22284 )
  37. Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 (Thermo Fisher Scientific, Invitrogen, catalog number: R37116 )
  38. DAPI Solution (1 mg/ml) (Thermo Fisher Scientific, catalog number: 62248 )
  39. Prolong® Diamond Antifade Mountant (Thermo Fisher Scientific, Invitrogen, catalog number: P36961 )
  40. NIH3T3 media (see Recipes)
  41. NIH3T3 serum/antibiotic-free media (see Recipes)
  42. 0.2 M Dibasic Sodium Phosphate solution (see Recipes)
  43. 0.2 M Monobasic Sodium Phosphate solution (see Recipes)
  44. Modified electron microscopy fixative (MEM-fix) (see Recipes)
  45. Permeabilization buffer (see Recipes)
  46. PBGT (see Recipes)
  47. Primary antibody solution (see Recipes)
  48. Secondary antibody solution (see Recipes)

Equipment

  1. Eppendorf Research Plus single channel pipette, 100-1,000 µl (Eppendorf, catalog number: 3123000063 )
  2. Eppendorf Research Plus single channel pipette, 20-200 µl (Eppendorf, catalog number: 3123000055 )
  3. Eppendorf Research Plus single channel pipette, 0.5-10 µl (Eppendorf, catalog number: 3123000020 )
  4. Eppendorf Research Plus single channel pipette, 0.1-2.5 µl (Eppendorf, catalog number: 3123000012 )
  5. Allegra X-12R centrifuge (Beckman Coulter, model: Allegra® X-12R , catalog number: 392302)
  6. Heracell VIOS 160i CO2 incubator (at 37 °C and 5% CO2), (Thermo Fisher Scientific, model: HeracellTM VIOS 160i, catalog number: 51030287 )
  7. AE50 analytical balance (Mettler-Toledo International, model: AE50 )
  8. Aspirator
  9. Precision dual-chamber water bath 288 (Thermo Fisher Scientific, catalog number: 2853
  10. Biological safety cabinet (The Baker Company, catalog number: B40-112
  11. Cell counting chamber (Hausser Scientific, catalog number: 3200 )
  12. FisherbrandTM Fine Point High Precision Forceps (Fisher Scientific, catalog number: 22-327379 )
  13. Confocal laser-scanning microscope (Leica Microsystems, model: Leica TCS SP8 )

Software

  1. Leica Application Suite X (used to generate Tiffs)

Procedure

  1. Day 1: Seeding cells (see Note 1)
    Seed 0.5 x 106 NIH3T3 cells into 1.5 ml media per well of a 6-well cell culture dish. Use one well per experimental condition. If not performing transfection, begin at Step C1.
    Note: All steps dealing with cells should be performed within a biological safety cabinet until ready to begin immunocytochemistry.

  2. Day 2: Transfections (see Note 2)
    1. Adherent cells should be at a confluence of 50-80% prior to transfection. Remove media and replace with 1 ml pre-warmed (37 °C) serum and antibiotic-free growth media to maximize transfection efficiency.
    2. Transfect each well with a total of 2 μg plasmid DNA encoding protein of interest according to transfection reagent protocol (we use Lipofectamine 3000 with P3000).
    3. Aspirate the media of transfected well(s) 6 h post transfection, and replace with 1.5 ml complete media.

  3. Day 3: Re-seeding cells (see Note 3)
    1. In the biological safety cabinet clean 12 mm cover slips in 70% ethanol. Place the clean coverslips in individual wells of a 24-well cell culture plate. Apply UV light to coverslips in the open 24-well plate for 15 min to sterilize prior to re-seeding cells (see Note 4).
    2. Aspirate the media from well(s) of 6-well plate and wash NIH3T3 cells with pre-warmed (37 °C) DPBS. Aspirate DPBS from well(s).
    3. Add 500 μl of pre-warmed 37 °C trypsin to each well, and place in an incubator for 3-5 min until cells detach. Add 2 ml media to neutralize trypsin, and count cells in counting chamber.
    4. Transfer cell suspension to a 15 ml centrifuge tube, and centrifuge at ~200 x g for 5 min at room temperature.
    5. While cells are in the centrifuge, add 500 μl media to each coverslip-containing well of the 24-well plate.
    6. Aspirate the cell supernatant and resuspend cell pellet in 5 ml pre-warmed media.
    7. Add an appropriate volume of cell suspension so that each well with a coverslip contains 0.1 x 106 cells.
    8. Allow 6-8 h for cells to fully attach to the coverslip and allow for cytoneme regrowth, or optimally wait overnight (> 10 h) for full attachment and outgrowth (see Note 5).

  4. Day 4: Immunocytochemistry
    Crucial: From this point on, do not aspirate. Gently pipette when adding or removing any solution. Attempt to move the plate as little as possible, do not agitate plate during washes. This may fragment or break cytonemes. During incubations and washes, let solutions sit on cells without agitation.
    1. Pipette off media and wash cells quickly 3 times with room temperature DPBS.
    2. Remove DPBS and add 400 μl of MEM-fix to cells, then incubate for 7 min at room temperature.
    3. Pipette off the MEM-fix and wash the cells three times in DPBS at room temperature, for 5 min per wash.
    4. While cells are being washed in DPBS, prepare permeabilization buffer. See Recipes for instructions.
    5. Remove last DPBS wash, and add 400 μl of permeabilization buffer to each well and incubate at room temperature for 1 h.
    6. While cells are permeabilizing/blocking, prepare primary antibody dilutions in PBGT. See recipes for instructions.
    7. Remove permeabilization buffer, and add 300 μl of primary antibody dilution to each well. Incubate the plate overnight at 4 °C.

  5. Day 5: Secondary antibodies and cover slip mounting
    1. Prepare 50 ml of PBGT. See Recipes for instructions.
    2. Remove primary antibody dilution, and wash the cells three times in 400 μl of PBGT at room temperature for 5 min per wash.
    3. While the cells are being washed, dilute secondary antibodies, phalloidin and DAPI in PBGT. See Recipes for instructions.
    4. Remove PBGT, and add 300 μl of secondary antibody dilutions to each well. Incubate plate at room temperature for 1 h in the dark or wrapped in aluminum foil (see Note 6).
    5. Remove secondary antibody dilution, and wash the cells three times in 400 μl of PBGT at room temperature for 5 min per wash. Keep the plate in the dark during washes.
    6. Remove final PBGT wash, and add 500 μl distilled water to each well to allow easier removal of coverslips and dilute salt content. Use forceps to gently remove coverslip from well. Remove any excess liquid from the edge of the coverslip by dabbing on a Kimwipe. Mount coverslips by gently placing them on a single drop of ProLong diamond applied to the slide. Avoid applying pressure or disturbing coverslip until ProLong diamond has cured.
    7. Let slide cure overnight in the dark at room temperature, and image on inverted confocal microscope within 2 weeks (see Notes 7 and 8).

Data analysis

It is important to note the differences between cytonemes and other cellular projections when analyzing images, as this protocol also preserves stress fibers. We classify cytonemes in a cultured system as positive for signaling proteins, actin positive, and ~10 μm in length or greater, roughly 200 nm in diameter, and not originating from the basal surface of the cell that is in contact with the coverslip. This final criterion is important for the specific identification of cytonemes because cell adhesion sites including focal adhesions retain stress fibers along the basal surface of the cell. Stress fibers tend to be much more linear than cytonemes, and are typically observed to be attached to the surface of the coverslip (Vallenius, 2013). Cytonemes can form at any position along the cell and have dynamic projections in three dimensions (Figures 2 and 3). It is important to distinguish the differences between these two projections when quantifying cytonemes.


Figure 2. Maximum intensity projection (MIP) allows for accurate representation of cytoneme projections. A and B. NIH3T3 cell transfected with Shh. A. Single section NIH3T3 cell. Cytoneme position drifts in and out of focal plane and appears completely absent of F-actin (red). B. MIP encompassing the range of cytonemes in the same cell. Continuous cytoneme seen for both Shh (green) and F-actin channels. Scale bars = 25 µm.


Figure 3. MEM-fix preserves stress fibers and cytonemes. A-C. C57BL/6 MEF cell transfected with Shh. A. MIP of entire cell shows many cellular projections, arrow heads show stress fibers, while arrows highlight cytonemes. B. Basal section of the cell shows stress fibers radiating out of cell body in all directions. Arrow head stress fibers still present, while arrow cytonemes absent. Some cytonemes will also be present in basal section. C. MIP of the cell excluding the basal section attached to the coverslip. Cytonemes are seen orienting to adjacent cells. Arrow head stress fibers absent, arrow cytonemes present. Scale bar = 25 µm.

Notes

  1. The procedure provided has been tailored for NIH3T3 cells, but can be applied to a wide range of different cell lines in cultured systems (as in Figure 1). We have applied this general protocol format to multiple cell lines with success in preserving and imaging cytonemes, even when altering culture methods for specific cell line requirements. This includes Mouse Embryonic Fibroblasts (MEF), Inner medullary collecting duct (IMCD3), HEK293T, IMR-32, and SK-N-SH cells.
  2. We transfected our cell with Lipofectamine 3000. To maximize transfection efficiency, plasmid DNA, P3000, and Lipofectamine were incubated in Opti-mem media, then and added to serum and antibiotic free NIH3T3 media. Cells can be transfected with any plasmid DNA and preferred transfection reagent under optimized conditions.
  3. It should be noted that cell re-seeding is not always necessary after transfection. However, in our experience it allows for much cleaner immunocytochemistry imaging, as it removes any dead cells and excess particles that may have accumulated on the coverslips from first seeding of the cells (Figure 4). This protocol has very mild washing steps and does not remove much debris. Trypsinizing transfected cells and re-seeding becomes more important when imaging cytonemes that end up attaching to the coverslip itself.


    Figure 4. Re-seeding cells after transfections reduces nonspecific particle fluorescence and cellular debris. A and B. C57BL/6 MEF cells transfected with Shh. A. MEF cells seeded onto coverslip, transfected, and then stained with anti-Shh (green), phalloidin to mark F-actin (red) and DAPI (blue). B. MEF cells seeded, transfected, then trypsinized and re-seeded onto coverslip and stained with anti-Shh, phalloidin and DAPI. Scale bars = 25 µm.

  4. Until comfortable with this protocol, it is recommended that you use a minimum of 2 coverslips per experimental condition.
  5. You may shorten the time period if working with suspension cell lines, or monitoring early cytoneme outgrowth.
  6. From this point on keep exposure of cells to direct light to a minimum.
  7. During image acquisition on the confocal microscope, it is recommended to take multiple Z-steps covering most of the cell. Cytonemes frequently project from various sections throughout the Z-axis of the cell, and may fall out of the focal plane of any single section. To ensure correct full cytoneme length, images representing cytonemes length and interactions with other cells should be shown as maximum intensity projections of the entire Z-range of the cytoneme.
  8. During image acquisition and identification of cytonemes, avoid imaging of projections that initiate along the base of the cell and remain in contact with the coverslip. This will prevent inclusion of stress fibers in images.

Recipes

  1. NIH3T3 media
    DMEM (4.5 g/L glucose) is supplemented with 10% BCS, ~1% MEM NEAA, ~1% L-Glutamine, ~1% Pen/Strep, and ~1% sodium pyruvate
    To make ~550 ml NIH3T3 media, add:
    500 ml DMEM
    50 ml HyCloneTM Cosmic CalfTM Serum (BCS)
    5 ml MEM NEAA
    5 ml L-Glutamine
    5 ml Pen/Strep
    5 ml Sodium Pyruvate to 500 ml StericupTM
    Vacuum filter contents into receiver bottle and store at 4 °C for up to a month
  2. NIH3T3 serum/antibiotic-free media
    DMEM (4.5 g/L glucose) is supplemented with ~1% MEM NEAA, ~1% L-Glutamine, and ~1% sodium pyruvate
    To make ~500 ml NIH3T3 media, add:
    500 ml DMEM
    5 ml MEM NEAA
    5 ml L-Glutamine
    5 ml Sodium Pyruvate to 500 ml StericupTM
    Vacuum filter contents into receiver bottle and store at 4 °C for up to a month
  3. 0.2 M Dibasic Sodium Phosphate solution
    To make 1 L of 0.2 M Dibasic Sodium Phosphate solution, dissolve 28.392 g of Dibasic Sodium Phosphate in 1 L of ultrapure water
  4. 0.2 M Monobasic Sodium Phosphate solution
    To make 1 L of 0.2 M Monobasic Sodium Phosphate solution, dissolve 23.995 g of Monobasic Sodium Phosphate in 1 L of ultrapure water
  5. Modified electron microscopy fixative (MEM-fix)
    MEM-fix contains 4% formaldehyde, 0.5% glutaraldehyde and 0.1 M Phosphate buffer (pH 7.4)
    To make 10 ml of MEM-fix:
    1. Mix 8.1 ml 0.2 M Dibasic Sodium Phosphate solution and 1.9 ml 0.2 M Monobasic Sodium Phosphate solution in 15 ml centrifuge tube
    2. Remove 3.125 ml solution, so remaining volume is 6.875 ml
    3. Add 2.5 ml 16% formaldehyde and 625 μl 8% glutaraldehyde
    4. Invert tube several times to mix and use immediately prior to fixation
  6. Permeabilization buffer
    Permeabilization buffer consists of 5% Normal Goat or Donkey Serum (adjust as necessary to accommodate primary antibodies to be used), and 0.1% Triton X-100, in a 1 mg/ml Sodium Borohydride solution dissolved in distilled water
    To make 10 ml of permeabilization buffer:
    1. Dissolve 10 mg Sodium Borohydride in 10 ml distilled water in a 15 ml centrifuge tube. Sodium Borohydride dissolved in water releases gas, so make sure the lid is ajar to prevent excess gas build up and internal pressure in the centrifuge tube
    2. Remove 600 μl solution, so 9.4 ml Sodium Borohydride solution remains
    3. Add 500 μl Normal Goat Serum, and 100 μl 10% Triton X-100 (diluted in DPBS)
    4. Invert tube several times to mix. Use immediately and do not store with centrifuge tube lid sealed
  7. PBGT
    PBGT consists of 5% Normal Goat Serum, 0.1% Tween-20 in DPBS
    To make 50 ml of PBGT, add and mix:
    47 ml of DPBS
    2.5 ml Normal Goat Serum
    0.5 ml 10% Tween-20 (diluted in DPBS) into a 50 ml centrifuge tube
    Note: PBGT can be stored at 4 °C for several days until ready to use.
  8. Primary antibody solution
    Primary antibody dilution is 1:100 for anti-Shh
    To make primary antibody solution for 2 wells of a 24 well plate, add 6 μl of anti-Shh to 600 μl of PBGT in a 1.5 ml microcentrifuge tube
    Note: Prepare and use primary antibody solution immediately prior to use.
  9. Secondary antibody solution
    Dilute all secondary antibodies at 1:1,000, DAPI solution at 1:2,000, and phalloidin at 1:400, all diluted in PBGT
    Note: Prepare and use secondary antibody solution immediately prior to use.

Acknowledgments

This work was supported by R01GM114049 and R35GM122546 to SKO, St. Jude Cancer Center support grant NCI P30 CA021765, and by ALSAC of St. Jude Children’s Research Hospital. This protocol was adapted from the MEM-fix protocol originally described in Bodeen et al. (2017). The authors declare no conflicts of interest or competing interests.

References

  1. Bacallao, R., Sohrab, S., and Phillips, C. (2006). Guiding principles of specimen preservation for confocal fluorescence microscopy. In: Handbook of Biological Confocal Microscopy. Springer, 311-325.
  2. Bodeen, W. J., Marada, S., Truong, A. and Ogden, S. K. (2017). A fixation method to preserve cultured cell cytonemes facilitates mechanistic interrogation of morphogen transport. Development 144(19): 3612-3624.
  3. Ramírez-Weber, F. A. and Kornberg, T. B. (1999). Cytonemes: cellular processes that project to the principal signaling center in Drosophila imaginal discs. Cell 97(5): 599-607.
  4. Sanders, T. A., Llagostera, E. and Barna, M. (2013). Specialized filopodia direct long-range transport of SHH during vertebrate tissue patterning. Nature 497(7451): 628-632.
  5. Stanganello, E., Hagemann, A. I., Mattes, B., Sinner, C., Meyen, D., Weber, S., Schug, A., Raz, E. and Scholpp, S. (2015). Filopodia-based Wnt transport during vertebrate tissue patterning. Nat Commun 6: 5846.
  6. Tagliaferro, P., Tandler, C. J., Ramos, A. J., Pecci Saavedra, J. and Brusco, A. (1997). Immunofluorescence and glutaraldehyde fixation. A new procedure based on the Schiff-quenching method. J Neurosci Methods 77(2): 191-197.
  7. Vallenius, T. (2013). Actin stress fibre subtypes in mesenchymal-migrating cells. Open Biol 3(6): 130001.

简介

培养细胞的免疫细胞化学是用于确定细胞结构内蛋白质的组成和定位的常用且有效的技术。 然而,传统的培养细胞固定和染色方案不能有效地保存培养的细胞色素,长期专门用于形态发生转运的丝状伪足。 结果,进行了有限的机械审讯以评估其监管。 我们开发了一种用于培养细胞的固定方案,该方案保留了细胞质,允许对内源性和过表达的蛋白质进行免疫荧光分析,这些蛋白质定位于脆弱的细胞结构。

【背景】Cytonemes被分类为薄的(~200nm直径)基于肌动蛋白的丝状伪足,长度超过2μm,可以转运形态发生素(Ramírez-Weber和Kornberg,1999)。这些信号结构首先在发育中的 Drosophila 翼成像盘中进行了详细分类和描述,随后在小鼠,小鸡和斑马鱼模型生物中进行了观察(Ramírez-Weber和Kornberg,1999; Sanders et al。,2013; Stanganello et al。,2015)。在大多数情况下,只有对过表达的荧光标记蛋白进行实时成像才能进行细胞色素检测。由于传统的固定方案未能保存这些脆弱的细丝,因此对培养细胞的细胞色素的检查受到限制。这些并发症一直是决定在发育和组织稳态期间驱动细胞色素形成和功能的细胞机制以及确定这些过程是否在疾病中被破坏的限制因素。

为了克服这些限制,我们开发了一种基于修饰电子显微镜固定剂(MEM-fix)的方案,该方案可以保留培养细胞的细胞质。 MEM-fix的使用允许通过传统的免疫荧光方案检测在filopodial结构中的内源和过表达的目的蛋白(Bodeen 等,,2017)。通过将戊二醛添加至标准4%多聚甲醛固定剂溶液的最终工作浓度0.5%来产生MEM-固定。由于其有效保存亚细胞结构的能力,包括戊二醛,其通常用于固定用于基于电子显微镜的研究的细胞。虽然戊二醛由于其自发荧光的倾向而不是免疫荧光显微镜的最佳固定剂,但我们确定向透化缓冲液中加入26.4 mM硼氢化钠足以缓解这种不良副作用(Tagliaferro 等。,1997; Bacallao et al。,2006)。不幸的是,由于戊二醛限制抗体渗透到细胞中,MEM-fix不利于细胞质或核蛋白的染色,因此应限于检测整合膜或近膜蛋白。

我们最近使用这种技术来检测Hedgehog(Hh)通过培养的 Drosophila 细胞和小鼠成纤维细胞的细胞因子转运(Bodeen et al。,2017)。在这里,我们提供了NIH3T3细胞中细胞色素成像的优化方案,并提供了其对其他培养的哺乳动物细胞系的适应性的实例(图1)。对标准细胞固定方法的MEM-固定方案修改允许可重复检测细胞质和基于免疫荧光的培养细胞中跨膜和膜邻近蛋白的染色。 MEM-fix还保留荧光标记蛋白的信号,因此不仅仅依赖于基于免疫检测的方法。


图1.与多聚甲醛固定相比,MEM-fix增加培养细胞中细胞因子的保存。 A和B.用Shh转染的HEK293T细胞。 A.用多聚甲醛固定的细胞显示一些基于肌动蛋白的突起,但不显示Shh(绿色)阳性细丝。 B.用MEM-fix固定的细胞含有许多细胞色素投射,标志着F-肌动蛋白(红色)和Shh的存在。比例尺=25μm。

关键字:MEM固定, 细胞导管, 丝状伪足, 显微镜观察, 免疫荧光, 形态发生素

材料和试剂

  1. SHARP ®精密阻隔技巧,适用于P-1000和Eppendorf1,000,1,250μl(Denville Scientific,目录号:P1126)
  2. SHARP ®精密阻隔技巧,适用于P-200,200μl(Denville Scientific,目录号:P1122)
  3. SHARP ®精密阻隔技巧,适用于P-20,20μl(Denville Scientific,目录号:P1121)
  4. SHARP ®精密阻隔技巧,P-2和P-10超长,10μl(Denville Scientific,目录号:P1096-FR)
  5. Gold Seal TM Rite-On TM Micro Slides(Thermo Fisher Scientific,目录号:3050-002)
  6. 12毫米显微镜盖玻片-1.5(Fisher Scientific,目录号:12-545-81)
  7. TPP ®离心管,容量50 ml,聚丙烯(TPP Techno Plastic Products,目录号:91050)
  8. TPP ®离心管,容量15 ml,聚丙烯(TPP Techno Plastic Products,目录号:91015)
  9. Stericup TM 无菌真空过滤装置500 ml(默克,产品目录号:SCGPU05RE)
  10. 24孔板(Corning,Falcon ®,目录号:353226) 
  11. 6孔板Nunclon TM Delta Surface(赛默飞世尔科技,目录号:140675)
  12. 专业Kimtech Science TM Kimwipes TM (KCWW,Kimberly-Clark,目录号:34155)
  13. 优质微量离心管:1.5 ml(Fisher Scientific,目录号:05-408-129)
  14. NIH3T3(ATCC,目录号:CRL-1658)
  15. 超纯水
  16. DMEM(1x)4.5g / L D-葡萄糖,[ - ] L-谷氨酰胺,[ - ] HEPES,[ - ]丙酮酸钠(Thermo Fisher Scientific,Gibco TM ,目录号:11960044)< br />
  17. Opti-MEM TM 还原血清培养基(赛默飞世尔科技,目录号:31985070)
  18. 笔/ Strep,100x(默克,目录号:TMS-AB2-C)
  19. HyClone TM Cosmic Calf TM 血清(BCS)(GE Healthcare,目录号:SH30087.03)
  20. MEM NEAA(100x)MEM非必需氨基酸(赛默飞世尔科技,Gibco TM ,目录号:11140050)
  21. L-谷氨酰胺(100x)(100 ml)(Thermo Fisher Scientific,Invitrogen TM ,目录号:25030081)
  22. 丙酮酸钠(100 mM)100x(Thermo Fisher Scientific,Gibco TM ,目录号:11360070)
  23. Lipofectamine ® 3000转染试剂盒(Thermo Fisher Scientific,Invitrogen TM ,目录号:L3000-015)
  24. 70%(体积)乙醇在水中稀释
  25. 0.05%胰蛋白酶0.53 nM EDTA,1x [ - ]碳酸氢钠(Corning,目录号:25-052-Cl)
  26. DPBS,1x(Dulbecco's Phosphate-Buffered Saline)(Corning,Cellgro TM ,目录号:21-031-CM)
  27. 磷酸氢二钠(Sigma-Aldrich,目录号:S0876)
  28. 磷酸二氢钠(Sigma-Aldrich,目录号:S5011)
  29. 硼氢化钠(Sigma-Aldrich,目录号:213462-25G)
  30. 甲醛,16%,不含甲醇,超纯EM等级(Polysciences,目录号:18814-10)
  31. 戊二醛,8%水溶液,EM级(电子显微镜科学,目录号:16019)
  32. 普通山羊血清(10 ml)(Jackson ImmunoResearch,目录号:005-000-121)
  33. Triton X-100(Sigma-Aldrich,目录号:T9284)
  34. Tween 20(Acros Organics,目录号:AC233360010)&nbsp;
  35. Sonic hedgehog(Shh)抗体(H-160),兔多克隆抗体(Santa Cruz Biotechnology,目录号:sc-9024)
  36. Alexa Fluor TM 633鬼笔环肽(赛默飞世尔科技,目录号:A22284)
  37. 山羊抗兔IgG(H + L)二抗,Alexa Fluor 488(Thermo Fisher Scientific,Invitrogen,目录号:R37116)
  38. DAPI溶液(1mg / ml)(Thermo Fisher Scientific,目录号:62248)
  39. 延长®金刚石抗褪色剂(Thermo Fisher Scientific,Invitrogen,目录号:P36961)
  40. NIH3T3培养基(见食谱)
  41. NIH3T3血清/无抗生素培养基(见食谱)
  42. 0.2 M二元磷酸钠溶液(见食谱)
  43. 0.2 M一元磷酸钠溶液(见食谱)
  44. 改良电子显微镜固定剂(MEM-fix)(见食谱)
  45. 透化缓冲液(见食谱)
  46. PBGT(见食谱)
  47. 一抗溶液(见食谱)
  48. 二抗溶液(见食谱)

设备

  1. Eppendorf Research Plus单通道移液器,100-1,000μl(Eppendorf,目录号:3123000063)
  2. Eppendorf Research Plus单通道移液器,20-200μl(Eppendorf,目录号:3123000055)
  3. Eppendorf Research Plus单通道移液器,0.5-10μl(Eppendorf,目录号:3123000020)
  4. Eppendorf Research Plus单通道移液器,0.1-2.5μl(Eppendorf,目录号:3123000012)
  5. Allegra X-12R离心机(Beckman Coulter,型号:Allegra ® X-12R,目录号:392302)
  6. Heracell VIOS 160i CO 2 培养箱(37°C和5%CO 2 ),(Thermo Fisher Scientific,型号:Heracell TM VIOS 160i ,产品目录号:51030287)
  7. AE50分析天平(Mettler-Toledo International,型号:AE50)
  8. 吸气器
  9. 精密双室水浴288(赛默飞世尔科技,目录号:2853)&nbsp;
  10. 生物安全柜(The Baker Company,目录号:B40-112)&nbsp;
  11. 细胞计数室(Hausser Scientific,目录号:3200)
  12. Fisherbrand TM Fine Point高精度镊子(Fisher Scientific,目录号:22-327379)
  13. 共聚焦激光扫描显微镜(Leica Microsystems,型号:Leica TCS SP8)

软件

  1. Leica Application Suite X(用于生成Tiff)

程序

  1. 第1天:播种细胞(见注1)
    将0.5×10 6个 6 NIH3T3细胞接种到6孔细胞培养皿的每孔1.5ml培养基中。每个实验条件使用一个孔。如果不进行转染,请从步骤C1开始。
    注意:所有处理细胞的步骤都应在生物安全柜内进行,直到准备开始免疫细胞化学。

  2. 第2天:转染(见注2)
    1. 在转染之前,贴壁细胞应处于50-80%的汇合处。取出培养基,换上1 ml预热(37°C)血清和无抗生素的生长培养基,以最大限度地提高转染效率。
    2. 根据转染试剂方案(我们使用含有P3000的Lipofectamine 3000),用总共2μg编码目的蛋白的质粒DNA转染每个孔。
    3. 转染后6小时吸出转染孔的培养基,并用1.5ml完全培养基替换。

  3. 第3天:重新播种细胞(见注3)
    1. 在生物安全柜中,清洁12毫米的盖子在70%乙醇中滑动。将干净的盖玻片放入24孔细胞培养板的各个孔中。在重新接种细胞之前,将紫外线照射到开口24孔板中的盖玻片上15分钟进行灭菌(见注4)。
    2. 从6孔板孔中吸出培养基,用预热(37℃)DPBS洗涤NIH3T3细胞。从井中吸取DPBS。
    3. 每孔加入500μl预热的37°C胰蛋白酶,置于培养箱中3-5分钟,直至细胞脱落。加入2 ml培养基中和胰蛋白酶,计数细胞室中的细胞。
    4. 将细胞悬浮液转移到15 ml离心管中,在室温下以~200 x g 离心5分钟。
    5. 当细胞在离心机中时,在24孔板的每个含盖玻片的孔中加入500μl培养基。
    6. 吸出细胞上清液,将细胞沉淀重悬于5 ml预热培养基中。
    7. 加入适量的细胞悬液,使每个带盖玻片的孔含有0.1×10 6 细胞。
    8. 允许细胞完全附着到盖玻片上6-8小时并允许细胞色素再生,或者最佳等待过夜(> 10小时)以完全附着和生长(参见注释5)。

  4. 第4天:免疫细胞化学
    至关重要:从现在开始,不要吸气。添加或移除任何溶液时轻轻移液。尝试尽可能少地移动板,在洗涤过程中不要搅拌板。这可能会破坏或破坏细胞质。在孵育和洗涤过程中,让溶液静置在细胞上。
    1. 移取培养基并用室温DPBS快速洗涤细胞3次。
    2. 取出DPBS并向细胞中加入400μlMEM-fix,然后在室温下孵育7分钟。
    3. 移取MEM-fix并在室温下在DPBS中洗涤细胞三次,每次洗涤5分钟。
    4. 在DPBS中洗涤细胞时,准备透化缓冲液。有关说明,请参阅食谱。
    5. 去除最后一次DPBS清洗,每孔加入400μl透化缓冲液,室温孵育1小时。
    6. 当细胞透化/阻断时,在PBGT中制备一抗稀释液。有关说明,请参阅食谱。
    7. 去除透化缓冲液,并向每个孔中加入300μl一抗稀释液。将板在4°C孵育过夜。

  5. 第5天:二抗和盖玻片安装
    1. 准备50毫升PBGT。有关说明,请参阅食谱。
    2. 去除一抗稀释液,在室温下用400μlPBGT洗涤细胞三次,每次洗涤5分钟。
    3. 在洗涤细胞的同时,在PBGT中稀释二抗,鬼笔环肽和DAPI。有关说明,请参阅食谱。
    4. 去除PBGT,并向每个孔中加入300μl二抗稀释液。将板在室温下孵育1小时,避光或用铝箔包裹(见注6)。
    5. 去除二抗稀释液,并在室温下在400μlPBGT中洗涤细胞三次,每次洗涤5分钟。在洗涤过程中将盘子保持在黑暗中。
    6. 去除最终的PBGT洗涤液,并向每个孔中加入500μl蒸馏水,以便更容易地去除盖玻片并稀释盐含量。用镊子从孔中轻轻取下盖玻片。轻轻擦拭Kimwipe,从盖玻片边缘去除多余的液体。将盖玻片轻轻放在涂在载玻片上的一滴ProLong金刚石上,然后盖上盖玻片。在ProLong钻石固化之前,避免施加压力或打扰盖玻片。
    7. 在室温下在黑暗中滑动固化过夜,并在2周内在倒置共聚焦显微镜上成像(见注7和8)。

数据分析

重要的是要注意分析图像时细胞质和其他细胞投射之间的差异,因为该方案还保留了应力纤维。我们将培养系统中的细胞色素分类为信号蛋白阳性,肌动蛋白阳性,长度为~10μm或更大,直径约200nm,而不是源自与盖玻片接触的细胞的基底表面。该最终标准对于细胞内酯的特异性鉴定是重要的,因为包括粘着斑的细胞粘附位点沿着细胞的基底表面保留应力纤维。应力纤维往往比细胞色素更加线性,并且通常被观察到附着在盖玻片的表面上(Vallenius,2013)。 Cytonemes可以在细胞的任何位置形成,并具有三维动态投影(图2和3)。在量化细胞色素时,区分这两个投影之间的差异非常重要。


图2.最大强度投影(MIP)允许准确表示细胞质投射。 A和B.用Hh转染的NIH3T3细胞。 A.单节NIH3T3细胞。 Cytoneme位置在焦平面内外漂移,并且看起来完全没有F-肌动蛋白(红色)。 B. MIP包括同一细胞中的细胞色素范围。对Shh(绿色)和F-肌动蛋白通道均观察到连续的细胞色素。比例尺=25μm。


图3. MEM-fix可保留应力纤维和细胞色素。 A-C。用Shh转染的C57BL / 6 MEF细胞。 A.整个细胞的MIP显示许多细胞投射,箭头显示应力纤维,而箭头突出显示细胞质。 B.细胞的基底部分显示应力纤维在所有方向上从细胞体辐射出来。箭头头部应力纤维仍然存在,而箭头细胞膜不存在。一些细胞色素也将存在于基底部分。 C.细胞的MIP,不包括附着于盖玻片的基部。看到Cytonemes定向于相邻细胞。没有箭头头部应力纤维,箭头细胞质存在。比例尺=25μm。

笔记

  1. 所提供的程序是针对NIH3T3细胞定制的,但可以应用于培养系统中的各种不同细胞系(如图1所示)。我们已将这种通用方案形式应用于多种细胞系,成功地保存和成像细胞质,甚至在改变特定细胞系要求的培养方法时也是如此。这包括小鼠胚胎成纤维细胞(MEF),内髓集合管(IMCD3),HEK293T,IMR-32和SK-N-SH细胞。
  2. 我们用Lipofectamine 3000转染我们的细胞。为了最大化转染效率,将质粒DNA,P3000和Lipofectamine在Opti-mem培养基中培养,然后加入到血清和无抗生素的NIH3T3培养基中。在优化条件下,可以用任何质粒DNA和优选的转染试剂转染细胞。
  3. 应该注意的是,转染后并不总是需要细胞再接种。然而,根据我们的经验,它可以进行更清洁的免疫细胞化学成像,因为它可以去除任何死细胞和可能从细胞第一次接种后积累在盖玻片上的多余颗粒(图4)。该方案具有非常温和的洗涤步骤,并且不会去除太多碎屑。当对最终附着于盖玻片本身的细胞色素进行成像时,胰蛋白酶消化转染的细胞并重新播种变得更加重要。


    图4.转染后再接种细胞减少非特异性颗粒荧光和细胞碎片。 A和B.用Shh转染的C57BL / 6 MEF细胞。 A.将MEF细胞接种到盖玻片上,转染,然后用抗Shh(绿色),鬼笔环肽染色以标记F-肌动蛋白(红色)和DAPI(蓝色)。 B.接种MEF细胞,转染,然后用胰蛋白酶消化并重新接种到盖玻片上,并用抗Shh,鬼笔环肽和DAPI染色。比例尺=25μm。

  4. 在熟悉此协议之前,建议您在每个实验条件下使用至少2个盖玻片。
  5. 如果使用悬浮细胞系或监测早期的cytoneme生长,您可以缩短时间段。
  6. 从这一点开始,保持细胞暴露在最低限度。
  7. 在共聚焦显微镜上进行图像采集时,建议采用多个Z步骤覆盖大部分细胞。 Cytonemes经常从整个Z轴的各个部分投射,并且可能从任何一个部分的焦平面掉出来。为了确保正确的完整细胞色素长度,代表细胞质长度和与其他细胞相互作用的图像应显示为细胞质的整个Z-范围的最大强度投影。
  8. 在图像采集和识别细胞色素期间,避免成像沿着细胞基部起始并保持与盖玻片接触的突起。这样可以防止在图像中包含应力纤维。

食谱

  1. NIH3T3媒体
    DMEM(4.5 g / L葡萄糖)补充10%BCS,~1%MEM NEAA,~1%L-谷氨酰胺,~1%Pen / Strep和~1%丙酮酸钠
    要制作~550 ml NIH3T3培养基,请添加:
    500毫升DMEM
    50毫升HyClone TM Cosmic Calf TM 血清(BCS)
    5毫升MEM NEAA
    5毫升L-谷氨酰胺
    5毫升笔/ Strep
    5毫升丙酮酸钠至500毫升Stericup TM
    将内容物真空过滤到接收瓶中,并在4°C下储存长达一个月
  2. NIH3T3血清/无抗生素培养基
    DMEM(4.5 g / L葡萄糖)补充~1%MEM NEAA,~1%L-谷氨酰胺和~1%丙酮酸钠
    制作约500毫升NIH3T3培养基,添加:
    500毫升DMEM
    5毫升MEM NEAA
    5毫升L-谷氨酰胺
    5毫升丙酮酸钠至500毫升Stericup TM
    将内容物真空过滤到接收瓶中,并在4°C下储存长达一个月
  3. 0.2 M二元磷酸钠溶液
    制备1升0.2M磷酸氢二钠溶液,将28.392克磷酸氢二钠溶于1升超纯水中
  4. 0.2 M一元磷酸钠溶液
    制备1升0.2M磷酸二氢钠溶液,将23.995克一元磷酸钠溶于1升超纯水中
  5. 改良电子显微镜固定剂(MEM-fix)
    MEM-fix含有4%甲醛,0.5%戊二醛和0.1M磷酸盐缓冲液(pH 7.4)
    制作10毫升MEM-fix:
    1. 在15 ml离心管中混合8.1 ml 0.2 M磷酸氢二钠溶液和1.9 ml 0.2 M磷酸二氢钠溶液
    2. 去除3.125毫升溶液,剩余体积为6.875毫升
    3. 加入2.5毫升16%甲醛和625微升8%戊二醛
    4. 在固定前立即将管反复混合并立即使用
  6. 透化缓冲区
    透化缓冲液由5%正常山羊或驴血清组成(根据需要调整以适应所用的一抗)和0.1%Triton X-100,溶于蒸馏水中的1 mg / ml硼氢化钠溶液
    制作10毫升透化缓冲液:
    1. 将10mg硼氢化钠溶于15ml离心管中的10ml蒸馏水中。溶于水中的硼氢化钠释放出气体,因此确保盖子是半开的,以防止过量的气体积聚和离心管内的压力。
    2. 取出600μl溶液,保留9.4 ml硼氢化钠溶液
    3. 加入500μl普通山羊血清和100μl10%Triton X-100(用DPBS稀释)
    4. 反转管多次混合。立即使用,不要与离心管盖密封存放
  7. PBGT
    PBGT由5%正常山羊血清,0.1%吐温-20在DPBS中组成
    制作50毫升PBGT,加入并混合:
    47毫升DPBS
    2.5毫升普通山羊血清
    将0.5ml 10%Tween-20(在DPBS中稀释)加入50ml离心管中 注意:PBGT可以在4°C下储存数天,直到准备使用。
  8. 一抗解决方案
    抗Shh的一抗稀释度为1:100 为24孔板的2个孔制备一抗溶液,在1.5 ml微量离心管中加入6μl抗Shh至600μlPBGT
    注意:在使用前立即准备并使用一抗溶液。
  9. 二抗溶液
    稀释1:1,000的所有二抗,1:2,000的DAPI溶液和1:400的鬼笔环肽,均在PBGT中稀释
    注意:在使用前立即准备并使用二抗溶液。

致谢

这项工作得到了R01GM114049和R35GM122546对SKO,St. Jude癌症中心支持补助金NCI P30 CA021765以及St. Jude儿童研究医院的ALSAC的支持。该协议改编自最初在Bodeen 等人(2017)中描述的MEM-fix协议。作者声明没有利益冲突或竞争利益。

参考

  1. Bacallao,R.,Sohrab,S。和Phillips,C。(2006)。 共焦荧光显微镜样本保存的指导原则。 :生物共聚焦显微镜手册。施普林格,311-325。
  2. Bodeen,W.J.,Marada,S.,Truong,A。和Ogden,S.K。(2017)。 保存培养的细胞内分泌物的固定方法有助于对形态发生素转运进行机械询问。 发展 144(19):3612-3624。
  3. Ramírez-Weber,F。A.和Kornberg,T。B.(1999)。 Cytonemes:投射到 Drosophila imaginal主要信号中心的细胞过程光盘。 Cell 97(5):599-607。
  4. Sanders,T.A.,Llagostera,E。和Barna,M。(2013)。 专门的丝状伪足直接远程运输SHH脊椎动物组织模式。 自然 497(7451):628-632。
  5. Stanganello,E.,Hagemann,A.I。,Mattes,B.,Sinner,C.,Meyen,D.,Weber,S.,Schug,A.,Raz,E。和Scholpp,S。(2015)。 脊椎动物组织模式中基于Filopodia的Wnt转运。 Nat Commun 6:5846。
  6. Tagliaferro,P.,Tandler,C.J。,Ramos,A.J.,Pecci Saavedra,J。和Brusco,A。(1997)。 免疫荧光和戊二醛固定。基于席夫淬灭法的新程序。 J Neurosci Methods 77(2):191-197。
  7. Vallenius,T。(2013)。 肌动蛋白应力纤维亚型在间充质迁移细胞中。 Open Biol 3(6):130001。
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Hall, E. T. and Ogden, S. K. (2018). Preserve Cultured Cell Cytonemes through a Modified Electron Microscopy Fixation. Bio-protocol 8(13): e2898. DOI: 10.21769/BioProtoc.2898.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。