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TUNEL Assay in Kiwifruit Stigmatic Arms
奇异果柱头臂的TUNEL检测   

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Annals of Botany
Jul 2014

Abstract

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) is a method for detecting DNA fragmentation by labelling the 3' terminal end of nucleic acids. This method can be used both in animal and plant tissues. In animal tissues, the use of Proteinase K is sufficient for permeabilizing the cells and to obtain optimal labelling, but in plant tissues, the presence of the cell wall, does not allow proper labelling. For this reason, we carried out several modifications to the original TUNEL protocol (ApoAlert® DNA Fragmentation Assay Kit, Clontech) to obtain an optimal labelling. These modifications were additional treatments with cellulase, Triton X-100 and Proteinase K. Also, we describe the optimization of the positive controls by adjusting the units of DNase used. The PI concentration for counterstaining has been also specifically adjusted to avoid excessive background noise and hence to correctly observe both labeled and unlabelled nuclei. This work also describes an additional protocol to collect, store and include samples (specifically stigmatic arms) in such a way that they do not interfere with the TUNEL labelling.

Keywords: Programmed Cell Death (具有细胞死亡), TUNEL assay (tunel法), DNA fragmentation assay (DNA碎片检测), Kiwifruit (kiwifruit), Plant reproductive biology (植物生殖生物学)

Materials and Reagents

  1. Acetic acid (CH3COOH) (Panreac Applichem)
  2. Acetone [(CH3)2CO] (Panreac Applichem)
  3. ApoAlert® DNA Fragmentation Assay Kit (Clontech, catalog number: 630107 )
  4. (3-Aminopropyl) triethoxy-silane (APTES) (C9H23NO3Si) (Sigma-Aldrich, catalog number: 440140 )
  5. Cellulase Onozuka RS (Duchefa Biochemie, catalog number: C8003 )
  6. Citifluor Solid Mountant kit (Agar Scientific, catalog number: AGR1326 )
  7. Clear nail polish
  8. Deionized H2O (Milli-Q H2O)
  9. Distilled water
  10. Disodium hydrogen phosphate (Na2HPO4) (Duchefa Biochemie)
  11. Ethylenediaminetetraacetic acid (EDTA) (C10H16N2O8) (Duchefa Biochemie)
  12. Ethanol (C2H5OH) (Panreac Applichem)
  13. Formaldehyde (HCHO) (36.5-38%) (Panreac Applichem, catalog number: 48 131328 )
  14. Monobasic potassium phosphate (KH2PO4) (Duchefa Biochemie)
  15. Paraffin (Panreac Applichem, catalog number: 253211 )
  16. Paraformaldehyde [(HCHO)n] (Panreac Applichem, catalog number: 141451 )
  17. Potassium chloride (KCl) (Duchefa Biochemie)
  18. Propidium iodide (PI) (Sigma-Aldrich, catalog number: P4864 )
  19. Proteinase K (Sigma-Aldrich, catalog number: P4850 )
  20. Recombinant DNase I (RNase-free) (TAKARA BIO, catalog number: 2270A )
  21. Sodium chloride (NaCl) (Duchefa Biochemie)
  22. Sodium hydroxide (NaOH) (Panreac Applichem)
  23. Sulfuric acid (H2SO4) (Sigma-Aldrich)
  24. Tertiary butyl alcohol/2-Methyl-2-Propanol (TBA) [(CH3)3COH] (Panreac Applichem, catalog number: 141903 )
  25. Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl) [NH2C(CH2OH)3·HCl] (Duchefa Biochemie)
  26. Triton X-100 (Sigma-Aldrich)
  27. Xylene [C6H4(CH3)2] (Panreac Applichem)
  28. Fixing solution (see Recipes)
  29. TBA solutions (see Recipes)
  30. TBA:paraffin (see Recipes)
  31. Formulated water (see Recipes)
  32. APTES-coated slides (see Recipes)
  33. 1x PBS buffer (see Recipes)
  34. 4% formaldehyde in 1x PBS buffer (see Recipes)
  35. 2% Cellulase Onozuka in 1x PBS buffer (see Recipes)
  36. 0.5% Triton X-100 in 1x PBS buffer (see Recipes)
  37. Tris-HCl (see Recipes)
  38. EDTA (see Recipes)
  39. 100 mM Tris-HCl (see Recipes)
  40. Proteinase K solution (see Recipes)
  41. 1x DNase I buffer (see Recipes)
  42. 1,500 U/ml DNase I (see Recipes)
  43. PI solution (see Recipes)
  44. Anti-Fade reagent (see Recipes)

Equipment

  1. 15 ml tubes
  2. 100 ml darkened bottles
  3. 100 ml darkened glass bottles
  4. 100 ml glass flasks
  5. 200 ml glass Coplin jars
  6. 250 ml glass jars
  7. Autoclave
  8. Filters sterile Filtropur S 0.2 (SARSTEDT AG)
  9. Flat surface (wall tile)
  10. Forceps
  11. Fume hood
  12. Glass coverslips
  13. Glass-covered trays
  14. Laboratory oven
  15. Leica TCS-SP2 confocal microscope
  16. Leuckart's bars for paraffin block preparation (ICT, SL., catalog number: 101 20996051645 )
  17. Magnetic stirrer with heating plate
  18. Microtome (Shibuya optical)
  19. Microtome Blades Accu-Edge® low-profile (Sakura)
  20. Paint brush
  21. pH-meter
  22. Scalpel with sterile blades
  23. Slides

Software

  1. LCS Software for Leica TCS-SP2 confocal microscope

Procedure

  1. Flower collection
    We collect flowers from mature female vines of kiwifruit, Actinidia chinensis var deliciosa (A.Chev.) A.Chev.) cv. ʻHaywardʼ growing in a commercial orchard located in north-western Spain (Galicia) and operated by Kiwi Atlántico S. A.
    1. Prepare plant material immediately after collection by detaching petals, sepals and stamens using a scalpel.


      Figure 1. Plant material. A. Kiwifruit female flower. B. Female pistil (ovary 124 with stigmatic arms). C. A female stigmatic arm detached from pistil.

    2. Submerge completely five pistils (ovary and stigmatic arms) in a 100 ml glass flask containing 60 ml fixing solution for 24 h at 4 °C (William et al., 1999).
    3. Remove fixing solution, add 60 ml 75% ethanol. Store at 4 °C until use.

  2. Paraffin embedding stigmatic arms
    1. Put 5 stigmatic arms, each from a different flower, in a 15 ml tube.
    2. Add 5 ml of TBA V (recipe II) solution to the samples and incubate 3 h at room 
temperature.
    3. Remove TBA V, add 5 ml TBA IV to the samples, and incubate overnight at 
room temperature.
    4. Remove TBA IV, add 5 ml TBA III to the samples, and incubate for 70 min at 
room temperature.
    5. Remove TBA III, add 5 ml TBA II to the samples, and incubate for 80 min at 
room temperature.
    6. Remove TBA II, add 5 ml TBA I to the samples, and incubate for 100 min at 
room temperature.
    7. Remove TBA I, add 5 ml pure TBA to the samples, and incubate for 85 min at 
38 °C in a laboratory oven.
    8. Replace TBA with fresh TBA and incubate for 45 min at 38 °C in a laboratory 
oven.
    9. Replace TBA with fresh TBA and incubate overnight at 38 °C in a laboratory 
oven.
    10. Remove the TBA, add 5 ml TBA: paraffin (previously maintained at 60 °C in a 
laboratory oven) and incubate for 24 h at 60 °C in a laboratory oven.
    11. Replace TBA: Paraffin with fresh one and incubate for 24 h at 60 °C in a laboratory oven.

    12. Remove the TBA: Paraffin add 5 ml paraffin (previously maintained at 60 °C) to the samples and incubate for 7 h at 60 °C in a laboratory oven.

    13. Replace the paraffin with fresh paraffin and incubate for 5 days at 60 °C in a laboratory oven.

    14. Put a thin layer of liquid paraffin into Leuckart bar. Place the samples arranged into horizontal position before the paraffin solidifies and completely cover with additional liquid paraffin. Two samples per mold can be prepared. Allow solidify at least 3-4 h.

    15. Label the paraffin blocks indicating the stigmatic arm orientation, and store them at room temperature until use.

  3. Section preparation
    1. Trim the paraffin blocks with the help of a scalpel to obtain an optimal cutting surface including the sample with a small paraffin frame.
    2. Fix the small paraffin frame on the microtome chuck.
    3. Set the microtome blade and adjust the positions of the blade and the sample.
    4. Cut the paraffin block to obtain ribbons of 10 μm-thick sections containing serially sectioned stigmatic arms. Pick up the sections with forceps or paint brush and store them in a glass-covered tray.

    5. Put a few drops of formulated water on the surface of previously prepared APTES-coated slides.

    6. Pick up the sections containing stigmatic arm samples from the trays with the aid of a forceps and put them onto the surface of formulated water on the APTES-coated slides (about 15-18 sections per slide).

    7. Leave the sections overnight at 30 °C in a laboratory oven to allow the thin sections be expanded and fully attached to the slides.
    8. Store slides at room temperature.

  4. Sample preparation for microscopic detection
    This procedure is carried out using the ApoAlert® DNA Fragmentation Assay Kit. We usually handle four slides at the same time in each protocol session. From them, two slides contain experimental samples, and the other two are controls, one negative and the other positive. For this later one (positive control), the use of dedicated laboratory material (glass coplin jar, wall tile, forceps, etc.) is mandatory.
    Steps from two to eleven, and from twenty-six to twenty-nine follow the ApoAlert® DNA Fragmentation Assay Kit protocol (steps 1-10, and 12-14, respectively).
    1. Incubate the slides at 60 °C in a laboratory oven for 7 min to ensure the complete removal of paraffin.
    2. Remove paraffin by immersing the slides in Coplin jars containing fresh xylene. Incubate at room temperature for 5 min.

    3. Repeat once the second step by transferring the slides to a new Coplin jar containing fresh xylene.
    4. Immerse the slides in Coplin jars containing fresh 100% ethanol and incubate at room temperature for 5 min.

    5. Rehydrate the slides by sequentially incubating them for 3 min steps at room temperature in different Coplin jars containing: 100% ethanol 95% ethanol 85% ethanol 70% ethanol 50% ethanol.
    6. Immerse the slides in Coplin jars containing fresh 0.85% NaCl (p/v) and incubate them at room temperature for 5 min.

    7. Immerse the slides in Coplin jars containing fresh 1x PBS buffer and incubate them at room temperature for 5 min.
    8. Fix the slides by immersing them in Coplin jars containing fresh 4% formaldehyde and incubate them at room temperature for 15 min.

      Note: Let cellulase to thaw on ice to be used in step 12.

    9. Wash the slides by immersing them in Coplin jars containing 1x PBS buffer, and incubate them at room temperature for 5 min.
    10. Transfer the slides to another Coplin jar containing 1x PBS buffer, and incubate them again at room temperature for 5 min.
    11. Allow the liquid to drain thoroughly and place the slides on a flat surface (wall tile). The cellulase, Triton X-100 and Proteinase K treatments must be carried out with care due to risk of losing sections, which can slip away from the slides.
    12. Cover every section with 2% cellulase Onozuka RS. Solution must completely cover the sections. Usually this can be done with 1 ml of solution per slide.
    13. Cover the slides with inverted Coplin jars containing wet paper towels in the bottom to ensure high humidity (Figure 2).


      Figure 2. Inverted Coplin jars containing wet paper towels in the bottom to ensure high humidity during laboratory oven incubation of slides

    14. Incubate at 37 °C for 90 min in a laboratory oven.
      Note: Prepare 0.5 % Triton X-100 in 1x PBS buffer to be used in step 18.
    15. Immerse the cellulase-treated slides in Coplin jars containing 1x PBS buffer and incubate them at room temperature for 5 min.
    16. Repeat twice the step 15 using a new Coplin jar containing fresh 1x PBS buffer.
    17. Allow the liquid to drain thoroughly and place the slides on a wall tile.
    18. Cover every section with 0.5% Triton X-100 in 1x PBS buffer. Solution must completely cover the sections. Usually this can be done with 1 ml of solution per slide.
    19. Incubate for 20 min at room temperature.
      Note: Prepare the 20 μg/ml Proteinase K solution to be used in step 23.
    20. Immerse the Triton X-100-treated slides in Coplin jars containing 1x PBS buffer and incubate them at room temperature for 5 min.

    21. Repeat twice the step 20 using a new Coplin jar containing fresh 1x PBS buffer.

    22. Allow the liquid to drain thoroughly and place the slides on a wall tile.

    23. Cover every section with the 20 μg/ml Proteinase K. Solution must completely cover the sections. Usually this can be done with 1 ml of solution per slide.
    24. Cover the slides with inverted Coplin jars containing wet paper towels in the bottom to ensure high humidity (Figure 2).
    25. Incubate at 37 °C for 30 min in a laboratory oven.
    26. Immerse the Triton X-100-treated slides in Coplin jars containing 1x PBS buffer and incubate them at room temperature for 5 min.
    27. Immerse the slides in Coplin jars containing 4% formaldehyde and incubate them at room temperature for 5 min.
    28. Repeat step 26 using a new Coplin jar containing fresh 1x PBS buffer.
    29. Leave the slides in fresh 1x PBS buffer, except that used to prepare the positive control.
    30. Prepare the positive control (with DNase) taking the slide containing the chosen sections for it and kept it separately from the other slides (experimental samples and negative control) after this point. It is necessary to use dedicated laboratory material (Coplin jars, forceps, wall tile, etc).
      1. Allow the liquid to drain thoroughly and place the slide on a wall tile.
      2. Add 1x DNase I buffer. Solution must completely cover the sections. Usually this can be done with 100 μl of solution.
      3. Incubate the slides at room temperature for 5 min.
      4. Gently tap the slides to remove liquid.
      5. Add 75 μl per slide of DNase I buffer containing 1,500 U/ml DNase I, as 
suggested in the In situ Cell Death Detection Kit, Fluorescein. Ensure that 
buffer completely covers the sections.
      6. Cover the slides with inverted Coplin jars containing wet paper towels in the 
bottom to ensure high humidity (Figure 2).
      7. Incubate at 37 °C for 20 min in a laboratory oven.
      8. Gently tap the slide to remove the liquid.
      9. Immerse the slide in Coplin jars containing 1x PBS buffer for 5 min twice.

  5. Staining for TUNEL detection by microscopy (using the ApoAlert® DNA Fragmentation Assay Kit)
    This procedure is carried out using the ApoAlert® DNA Fragmentation Assay Kit using all samples (experimental, negative and positive samples). Remember to keep the positive control separately from the other slides.
Steps from one to twelve follow the ApoAlert® DNA Fragmentation Assay Kit protocol (steps 1-5, 7-9, 11-16).
    1. Remove the slides from the 1x PBS buffer and tap gently to remove excess liquid; place the slides on a wall tile.
    2. Cover the slides with 100 μl of equilibration buffer using a micropipette.

    3. Using forceps, gently place a piece of plastic coverslip on top of the slides to evenly spread the liquid.

    4. Equilibrate at room temperature for 10 min. Thaw Nucleotide Mix on ice and prepare TdT incubation buffer for all samples and controls. Protect Nucleotide Mix and TdT incubation buffer from light. Keep on ice at all times. Protect the slides from light after staining.

      Use the following table (or similar) to prepare the experimental samples and positive controls:

      Component
      Volume
      Total No. of reactions
      Total volume
      Equilibration buffer
      45 μl
      noo fslides+1
      X μl
      Nucleotide mix
      5 μl
      noo fslides+1
      X μl
      TdT enzyme total
      1 μl
      noo fslides+1
      X μl
      Total
      51 μl
      noo fslides+1
      X μl

      Use the following table (or similar) to prepare the negative controls:

      Component
      Volume
      Total No. of reactions
      Total volume
      Equilibration buffer
      45 μl
      1
      X μl
      Nucleotide mix
      5 μl
      1
      X μl
      Milli-Q H2O
      1 μl
      1
      X μl
      Total
      51 μl
      1
      X μl

    5. Using forceps, remove the plastic coverslip and gently tap the slides to remove excess liquid. Manage with care due to risk of losing sections. Carefully blot dry around the edges with tissue paper and place the slides on a wall tile.

    6. Gently place 50 μl of TdT incubation buffer on the slides with a micropipette.
    7. Using forceps, gently place a piece of plastic coverslip on top of the slides to evenly spread the liquid.
    8. To perform the tailing reaction, place the slides in darkness at 37 °C for 60 min in a laboratory oven. Cover the slides with inverted Coplin jars containing wet paper towels in the bottom to ensure high humidity and maintain in darkness by covering with aluminium foil.
    9. Using forceps, remove the plastic coverslips. Manage with care due to risk of losing sections.
    10. Terminate the tailing reaction by immersing the slides in Coplin jars containing 2x SSC buffer. Incubate at room temperature for 15 min.
    11. Wash the slides by immersing them in fresh Coplin jars containing 1x PBS buffer. Incubate at room temperature for 5 min.
    12. Repeat twice the step 11 using a new Coplin jar containing fresh 1x PBS buffer.
      Note: Prepare 0.5 μg/ml PI to be used in step 14.

    13. Allow the liquid to drain thoroughly and place the slides on a wall tile.

    14. Cover every section with 0.5 μg/ml PI. Solution must completely cover the sections. Usually this can be done with 1 ml of solution per slide.

    15. Incubate at room temperature for 5 min.

    16. Wash the tissues by transferring the slides to fresh Coplin jars containing Milli-Q H2O and incubate at room temperature for 5 min.
    17. Repeat step 16 twice.
      Note: Prepare the Anti-Fade reagent to be used in step 19.

    18. Allow the liquid to drain thoroughly and place the slides on a wall tile.

    19. Add 150 μl of Anti-Fade reagent per slide and cover the treated portion of the slide containing the sections with a glass coverslip.

    20. Seal the edges of the coverslip with clear nail polish and allow drying for at least 5-10 min. The samples can be immediately examined or stored overnight at 4 °C in darkness, for their observation just next day. We usually examine sections in a Leica TCS-SP2 confocal microscope. Fluorescence generated by incorporation of fluorescein-dUTP at the free 3ʼ-hydroxyl ends of fragmented DNA, is detected using an excitation wavelength of 488 nm for the TUNEL reaction and 561 nm for PI, with detection in the range of 492-550 nm and 581-625 nm, respectively.

Representative data

This improved protocol for TUNEL assay of DNA fragmentation in kiwifruit tissues relies on the use of an improved permeabilization of tissues by treatments with cellulase, Triton X-100, and adjusted Proteinase K, increased DNase I units (1,500 U/ml) for the generation of positive controls, and optimization of counterstaining with reduced IP concentration (0.5 μg/ml). These modifications allowed us to clearly visualize the differences between negative and positive nuclei for TUNEL (Figure 3A-C), in contrast with the results obtained using the original protocol of the ApoAlert® DNA Fragmentation Assay Kit, which was not specifically designed for plant tissues (Figure 3D-F). In the Figure 3A, TUNEL-positive nuclei can be observed as they are showing bright green fluorescence, while in the Figure 3B all present nuclei are stained in red. Finally, in the Figure 3C, all present nuclei can be observed, but those being TUNEL positive are stained in yellow whereas those TUNEL negative are stained in light red or orange. On the contrary, with the ApoAlert® kit (Figure 3D-F) since no TUNEL-positive nuclei can be observed (Figure 3D), all present nuclei can be observed in red (Figure 3E) or after counterstaining in orange (Figure 3F).


Figure 3. Comparison of TUNEL assays in positive controls of kiwifruit tissues. A-C. Positive control sections of kiwifruit stigmatic arms stained using the improved TUNEL protocol described here. D-F. Positive control sections of kiwifruit stigmatic arms stained using the ApoAlert® DNA Fragmentation Assay Kit (Clontech). A, D. Fluorescence detected at 488 nm for the observation of the TUNEL stained nuclei (arrows). B, E. Fluorescence detected at 561 nm for the observation of the IP stained nuclei (arrows). C, F. Counterstaining by simultaneous fluorescence detection at both wavelengths (488 and 561 nm) of stained nuclei with TUNEL and IP (arrows).

Recipes

  1. Fixing solution (100 ml)

    Ethanol:acetic acid (3:1, v/v)

    Mix 75 ml absolute ethanol with 25 ml acetic acid
  2. TBA solutions (100 ml)
    TBA I

    Pure TBA
    75 ml
    96% ethanol
    25 ml
    TBA II

    TBA I
    73.2 ml
    96% ethanol
    26.8 ml
    TBA III

    TBA II
    63.6 ml
    96% ethanol
    21.4 ml
    H2O
    15 ml
    TBA IV

    TBA III
    57 ml
    96% ethanol
    21.6 ml
    H2O
    21.4 ml
    TBA V

    TBA IV
    50 ml
    96% ethanol
    15 ml
    H2O
    35 ml
    TBA can solidify at room temperature
    If this occurs, warm it in a laboratory oven at 38 °C
    Stored in 100 ml darkened glass bottles at room temperature until use
  3. TBA:paraffin (3:1 v/v) (100 ml)
    Combine 25 ml liquid paraffin with 75 ml pure TBA
    The paraffin should be at 60 °C
    Mix should be liquid to use
    Store in 250 ml glass jar at room temperature until use
  4. Formulated water (100 ml)
    Combine 3 ml formaldehyde 36.5-38% with 97 ml distilled water
  5. APTES-coated slides
    Prepare 2% APTES in acetone (v/v)
    Immerse the slides in 2% APTES for 3 min at room temperature
    Immerse the slides in acetone for 2 min at room temperature
    Repeat twice
    Immerse the slides in distilled water for 1 min at room temperature
    Allow to air dry (at least 24 h)
    Stored at room temperature until use
  6. 1x PBS buffer (pH 7.4, 1 L)
    10x PBS buffer (pH 7.4, 1L)
    NaCl
    80 g
    137 mM
    KCl
    2.2 g
    3 mM
    Na2HPO4
    14.4 g
    10 mM
    KH2PO4
    2.4 g
    2 mM
    Dissolve reagents in 800 ml Milli-Q H2O
    Adjust pH
    Complete volume to 1 L
    Autoclave
    Combine 100 ml 10x PBS buffer and 900 ml Milli-Q H2O
  7. 4% formaldehyde in 1x PBS buffer (pH 7.4, 1 L)
    Dissolve 40 g of paraformaldehyde in 500 ml of 1x PBS buffer under a fume hood
    Heat the solution at 70 °C while stirring
    Add one or two drops of sulfuric acid to facilitate paraformaldehyde dilution
    Complete volume to 1 L
    Stored in 100 ml darkened bottles for a maximum of 1-2 weeks at 4 °C or indefinitely at -20 °C
  8. 2% Cellulase Onozuka in 1x PBS buffer (10 ml)
    Dissolve 0.2 g of cellulase in 6 ml of 1x PBS buffer
    Incubate with slow stirring overnight at 4 °C
    Complete volume to 10 ml, filter sterilize (0.2 μm)
    Stored at -20 °C until use
  9. 0.5% Triton X-100 in 1x PBS buffer (10 ml)
    Dissolve 50 μl of Triton X-100 in 9.95 ml of 1x PBS buffer
    Use the solution freshly prepared
  10. Tris-HCl (0.5 M, pH 8.0) (100 ml)
    Dissolve 7.87 g of Tris-HCl in 100 ml distilled water
    Adjust pH with NaOH
    Autoclave and store at room temperature
  11. EDTA (0.2 M, pH 8.0) (100 ml)
    Dissolve 5.84 g of EDTA in 100 ml distilled water
    Adjust pH with NaOH
    Autoclave and store at room temperature
  12. 100 mM Tris-HCl (pH 8.0), 50 mM EDTA (3 ml)
    Combine 2 ml Tris-HCl buffer (0.5 M, pH 8.0) with 1 ml EDTA (0.2 M, pH 8.0)
  13. Proteinase K solution (20 μg/ml) (1 ml)
    Combine 2 μl of 10 mg/ml Proteinase K with 998 μl of 100 mM Tris-HCl (pH 8.0), 50 mM EDTA
  14. 1x DNase I buffer (100 μl)
    Dilute 10 μl10x DNase I buffer with 90 μl Milli-Q H2O
  15. 1,500 U/ml DNase I (75 μl)
    Combine 45 μl Milli-Q H2O, 22.5 μl Recombinant DNase I and 7.5 μl 10x DNase I buffer
  16. PI solution (0.5 μg/ml in PBS buffer) (5 ml)
    Stock: 1 mg/ml PI: Dissolve 10 mg of PI in 10 ml 1x PBS buffer
    Store the stock at 4 °C in darkness
    0.5 μg/ml PI solution: 2.5 μl stock and 5 ml 1x PBS buffer
  17. Anti-Fade reagent (1 ml)
    Combine 900 μl PVA solution (Citifluor Solid Mountant kit) and 100 μl AF100 (Citifluor Solid Mountant kit)

Acknowledgments

This research was supported by the Xunta de Galicia (Regional Government of Galicia, Spain; project PGIDIT 04RAG291002PR). We thank Kiwi Atlántico S. A. for providing the plant material. We thank M. S. Costa for her expert photographic assistance, and B. Lueiro for her technical assistance. This is a contribution of the Interuniversity Research Group in Biotechnology and Reproductive Biology of Woody Plants (BioVitAc Research group, code 09IDI1705).

References

  1. ApoAlert® DNA Fragmentation Assay Kit User Manual (Clontech, catalog number: 630107)
  2. Ferradas, Y., Lopez, M., Rey, M. and Gonzalez, M. V. (2014). Programmed cell death in kiwifruit stigmatic arms and its relationship to the effective pollination period and the progamic phase. Ann Bot 114(1): 35-45.
  3. In Situ Cell Death Detection Kit, Fluorescein (Roche Diagnostics, catalog number: 11 684 795 910)
  4. Williams, J. H., Jr., Friedman, W. E. and Arnold, M. L. (1999). Developmental selection within the angiosperm style: using gamete DNA to visualize interspecific pollen competition. Proc Natl Acad Sci U S A 96(16): 9201-9206.

简介

末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)是通过标记核酸的3'末端来检测DNA断裂的方法。该方法可以在动物和植物组织中使用。在动物组织中,蛋白酶K的使用足以使细胞透化并获得最佳标记,但是在植物组织中,细胞壁的存在不允许适当的标记。因此,我们对原始TUNEL方案(ApoAlert DNA Fragmentation Assay Kit,Clontech)进行了几个修改,以获得最佳标记。这些修饰是用纤维素酶,Triton X-100和蛋白酶K的另外处理。此外,我们通过调节所用的DNase的单位描述阳性对照的优化。用于复染的PI浓度也被特别调节以避免过度的背景噪声,并因此正确地观察标记的和未标记的核。该工作还描述了以不干扰TUNEL标记的方式收集,存储和包括样品(特别是污点臂)的附加方案。

关键字:具有细胞死亡, tunel法, DNA碎片检测, kiwifruit, 植物生殖生物学

材料和试剂

  1. 乙酸(CH 3 COOH)(Panreac Applichem)
  2. 丙酮[(CH 3)2 CO](Panreac Applichem)
  3. ApoAlert DNA片段化测定试剂盒(Clontech,目录号:630107)
  4. (3-氨基丙基)三乙氧基硅烷(APTES)(C 9 H 23 NO 3 Si)(Sigma-Aldrich,目录号:440140 )
  5. Cellulase Onozuka RS(Duchefa Biochemie,目录号:C8003)
  6. Citifluor固体填充剂试剂盒(Agar Scientific,目录号:AGR1326)
  7. 清除指甲油
  8. 去离子H 2 O(Milli-Q H 2 O)
  9. 蒸馏水
  10. 磷酸氢二钠(Na 2 HPO 4)(Duchefa Biochemie)
  11. 乙二胺四乙酸(EDTA)(C 10 H 16 N 12 O 18)(Duchefa Biochemie)
  12. 乙醇(C 2 H 5 OH)(Panreac Applichem)
  13. 甲醛(HCHO)(36.5-38%)(Panreac Applichem,目录号:48131328)
  14. 磷酸二氢钾(KH 2 PO 4)(Duchefa Biochemie)
  15. 石蜡(Panreac Applichem,目录号:253211)
  16. 多聚甲醛[(HCHO)n](Panreac Applichem,目录号:141451)
  17. 氯化钾(KCl)(Duchefa Biochemie)
  18. 碘化丙啶(PI)(Sigma-Aldrich,目录号:P4864)
  19. 蛋白酶K(Sigma-Aldrich,目录号:P4850)
  20. 重组DNA酶I(不含RNase)(TAKARA BIO,目录号:2270A)
  21. 氯化钠(NaCl)(Duchefa Biochemie)
  22. 氢氧化钠(NaOH)(Panreac Applichem)
  23. 硫酸(H 2 SO 4)(Sigma-Aldrich)
  24. 叔丁醇/2-甲基-2-丙醇(TBA)[(CH 3 3)3 COH](Panreac Applichem,目录号:141903)
  25. 三(羟甲基)氨基甲烷盐酸盐(Tris-HCl)[NH 2 CH(CH 2 OH)3·HCl](Duchefa Biochemie) br />
  26. Triton X-100(Sigma-Aldrich)
  27. 二甲苯[C 6 H 4(CH 3)2](Panreac Applichem)
  28. 固定解决方案(参见配方)
  29. TBA解决方案(参见配方)
  30. TBA:石蜡(见配方)
  31. 配方水(见配方)
  32. APTES涂层幻灯片(见配方)
  33. 1x PBS缓冲液(见配方)
  34. 4%甲醛的1x PBS缓冲液(见配方)
  35. 2%纤维素酶Onozuka在1x PBS缓冲液中(参见配方)
  36. 0.5%Triton X-100的1x PBS缓冲液(参见配方)
  37. Tris-HCl(参见配方)
  38. EDTA(见配方)
  39. 100 mM Tris-HCl(参见配方)
  40. 蛋白酶K溶液(见配方)
  41. 1x DNA酶I缓冲液(见配方)
  42. 1,500 U/ml DNase I(参见配方)
  43. PI解决方案(参见配方)
  44. 抗褪色试剂(见配方)

设备

  1. 15 ml管
  2. 100 ml变黑的瓶子
  3. 100毫升变黑的玻璃瓶
  4. 100ml玻璃烧瓶中
  5. 200 ml玻璃Coplin罐
  6. 250毫升玻璃罐
  7. 高压灭菌器
  8. 过滤器无菌Filtropur S 0.2(SARSTEDT AG)
  9. 平面(墙砖)
  10. 镊子
  11. 通风橱
  12. 玻璃盖片
  13. 玻璃盖托盘
  14. 实验室炉
  15. Leica TCS-SP2共焦显微镜
  16. 用于石蜡块制备的Leuckart棒(ICT,SL。,目录号:10120996051645)
  17. 带加热板的磁力搅拌器
  18. 切片机(涩谷光学)
  19. 切片机刀片Accu-Edge ®低调(樱花)
  20. 油漆刷
  21. pH计
  22. 无菌刀片的手术刀
  23. 幻灯片

软件

  1. LCS软件用于Leica TCS-SP2共聚焦显微镜

程序

  1. 花卉收集
    我们从猕猴桃的成熟雌性葡萄树中收集花,猕猴桃猕猴桃 var deliciosa (A.Chev。)A.Chev。 'Hayward'生长在位于西班牙西北部(加利西亚)的商业果园,由KiwiAtlánticoS.A公司经营。
    1. 通过使用手术刀分离瓣,萼片和雄蕊,在收集后立即准备植物材料。


      图1.植物材料。A.猕猴桃雌花。 B.雌性雌蕊 (具有耻骨臂的卵巢124)。 C.一个女性耻辱手臂脱离   雌蕊。

    2. 淹没完全五雌蕊(卵巢和 支链臂)在含有60ml固定溶液的100ml玻璃烧瓶中   在4℃下培养24小时(William等人,1999)。
    3. 取出固定溶液,加入60 ml 75%乙醇。 储存于4°C直至使用。

  2. 石蜡包埋的胳膊
    1. 将5个耻骨臂,每个从不同的花,在15毫升管中。
    2. 向样品中加入5 ml TBA V(食谱II)溶液,并在室温下孵育3小时。
    3. 取出TBA V,加入5毫升TBA IV的样品,并在室温下孵育过夜。
    4. 除去TBA IV,向样品中加入5 ml TBA III,并在室温下孵育70分钟。
    5. 除去TBA III,向样品中加入5ml TBA II,并在室温下温育80分钟。
    6. 除去TBA II,向样品中加入5毫升TBA I,并在室温下孵育100分钟。
    7. 除去TBA I,向样品中加入5ml纯TBA,并在38℃下在实验室烘箱中孵育85分钟。
    8. 用新鲜TBA替换TBA,并在38℃下在实验室烘箱中孵育45分钟。
    9. 用新鲜TBA替换TBA,并在38℃下在实验室烘箱中孵育过夜。
    10. 取出TBA,加入5 ml TBA:石蜡(以前保持在60℃)   ℃,在实验室烘箱中)并在60℃在a中孵育24小时 实验室烤箱。
    11. 替换TBA:将石蜡与新鲜的石蜡一起在实验室烘箱中在60℃下孵育24小时。
    12. 去除TBA:石蜡加入5毫升石蜡(以前保持 在60℃),并在60℃下在实验室中孵育7小时 烤箱。
    13. 用新鲜石蜡替换石蜡,并在60℃下在实验室烘箱中孵育5天。
    14. 把一薄层液体石蜡放入Leuckart酒吧。 放置 样品在石蜡固化之前排列成水平位置   并完全覆盖额外的液体石蜡。 每个两个样本 模具可以制备。 允许固化至少3-4小时。
    15. 标记石蜡块,指示耻性臂取向,并将其存储在室温直到使用。

  3. 剖面准备
    1. 在手术刀的帮助下修整石蜡块以获得最佳   包括具有小石蜡框架的样品的切割表面
    2. 将小石蜡框架固定在切片机卡盘上。
    3. 设置切片机刀片并调整刀片和样品的位置。
    4. 切割石蜡块以获得10μm厚部分的带 包含连续分段的耻骨臂。 拿起部分 镊子或油漆刷,并将它们存储在玻璃盖的托盘中。
    5. 将几滴配制的水放在先前制备的APTES涂覆的载玻片的表面上。
    6. 从托盘中取出含有耻骨臂样品的切片   在镊子的帮助下,将它们放在配方的表面上 水涂覆在APTES涂覆的载玻片上(每个载玻片约15-18个切片)。
    7. 在30°C下在实验室烘箱中放置切片过夜以允许 薄的部分膨胀并完全附接到载玻片。
    8. 在室温下存储载玻片。

  4. 用于微观检测的样品制备
    该程序使用ApoAlert DNA片段化测定试剂盒进行。我们通常在每个协议会话中同时处理四个幻灯片。从他们,两个幻灯片包含实验样本,其他两个是控件,一个负和另一个正。对于后面的(阳性对照),必须使用专用的实验室材料(玻璃杯子,墙砖,镊子,等)。
    从2到11,以及从26到29的步骤遵循ApoAlert DNA片段化测定试剂盒协议(分别为步骤1-10和12-14)。
    1. 在实验室烘箱中在60℃下孵育载玻片7分钟,以确保完全去除石蜡
    2. 通过将载玻片浸入含有新鲜二甲苯的Coplin瓶中来除去石蜡。在室温下孵育5分钟。
    3. 重复一次第二步,将幻灯片转移到包含新鲜二甲苯的新Coplin罐
    4. 将玻片浸没在含有新鲜100%乙醇的Coplin罐中,并在室温下孵育5分钟。
    5. 通过顺序孵育它们3分钟的步骤,再水合幻灯片   在室温下在不同的Coplin罐中包含:100%乙醇 95%乙醇85%乙醇70%乙醇50%乙醇
    6. 将载玻片浸没在含有新鲜的0.85%NaCl(p/v)的Coplin罐中,并在室温下孵育5分钟。
    7. 将载玻片浸入含有新鲜1x PBS缓冲液的Coplin罐中,并在室温下孵育5分钟。
    8. 固定幻灯片通过将它们浸泡在Coplin瓶子含有新鲜的4% 甲醛,并在室温下孵育15分钟。
      注意:让纤维素酶在冰上融化以在步骤12中使用。
    9. 通过将载玻片浸入含有1×PBS缓冲液的Coplin瓶中来洗涤载玻片,并在室温下孵育5分钟。
    10. 将载玻片转移到另一个含有1x PBS缓冲液的Coplin罐中,并在室温下再次孵育5分钟。
    11. 让液体彻底排空,将幻灯片放在平面上   表面(墙砖)。 纤维素酶,Triton X-100和蛋白酶K. 治疗必须小心进行,由于损失部分的风险,   其可以从载玻片滑落。
    12. 覆盖每个部分 2%纤维素酶Onozuka RS。 解决方案必须完全覆盖部分。 通常这可以用1ml溶液/载玻片进行。
    13. 用倒置的Coplin瓶盖覆盖载玻片,底部带有湿纸巾,以确保高湿度(图2)。


      图2.倒置的Coplin罐中含有湿纸巾 底部保证高湿度,在实验室烘箱孵化 幻灯片

    14. 在实验室烘箱中在37℃孵育90分钟。
      注意:在1x PBS缓冲液中制备0.5%Triton X-100用于步骤18.
    15. 将纤维素酶处理的载玻片浸渍在含有1x的Coplin瓶中 PBS缓冲液中,并在室温下孵育5分钟。
    16. 重复两次步骤15使用一个新的Coplin罐,含有新鲜的1×PBS缓冲液。
    17. 让液体彻底排空,将幻灯片放在墙上的瓷砖上。
    18. 用0.5%Triton X-100在1×PBS缓冲液中覆盖每个切片。 解决方案必须完全覆盖部分。 通常这可以做到 每个载玻片1ml溶液。
    19. 在室温下孵育20分钟。
      注意:准备在步骤23中使用的20μg/ml蛋白酶K溶液。
    20. 将Triton X-100处理的载玻片浸入Coplin罐中 1×PBS缓冲液,并在室温下孵育5分钟。
    21. 重复两次步骤20使用一个新的Coplin罐,含有新鲜的1×PBS缓冲液。
    22. 让液体彻底排空,将幻灯片放在墙上的瓷砖上。
    23. 用20μg/ml蛋白酶K覆盖每个切片 完全覆盖部分。 通常这可以用1ml的 解决方案。
    24. 用倒置的Coplin瓶盖覆盖载玻片,底部带有湿纸巾,以确保高湿度(图2)。
    25. 在实验室烘箱中在37℃孵育30分钟
    26. 将Triton X-100处理的载玻片浸入Coplin罐中 1×PBS缓冲液,并在室温下孵育5分钟
    27. 将幻灯片浸没在含有4%甲醛的Coplin罐中,并在室温下孵育5分钟
    28. 重复步骤26,使用含有新鲜1x PBS缓冲液的新Coplin罐
    29. 将载玻片置于新鲜的1×PBS缓冲液中,除了用于制备阳性对照
    30. 准备阳性对照(用DNase)取幻灯片 包含为它选择的部分,并保持它分开 其他载玻片(实验样品和阴性对照) 点。 有必要使用专用的实验室材料(Coplin 罐,镊子,墙砖,等)。
      1. 让液体彻底排空,将幻灯片放在墙上的瓷砖上。
      2. 加入1x DNA酶I缓冲液。 解决方案必须完全覆盖部分。 通常这可以用100μl的溶液完成。
      3. 孵育玻片在室温下5分钟。

  5. 通过显微镜检查(使用ApoAlert DNA片段化测定试剂盒)进行TUNEL检测的染色
    使用所有样品(实验,阴性和阳性样品)使用ApoAlert DNA片段化测定试剂盒进行该程序。 记住要保持阳性对照与其他幻灯片分开。 从1到12的步骤遵循ApoAlert ® DNA Fragmentation Assay Kit协议(步骤1-5,7-9,11-16)。
    1. 从1x PBS缓冲液中取出载玻片,轻轻轻轻取出多余的液体; 将幻灯片放在墙砖上
    2. 使用微量移液管用100μl平衡缓冲液覆盖载玻片。
    3. 使用镊子,轻轻地放置一片塑料盖玻片在幻灯片的顶部,以均匀地分布液体。
    4. 在室温下平衡10分钟。 解冻核苷酸混合 冰并为所有样品和对照制备TdT孵育缓冲液。 保护核苷酸混合物和TdT孵育缓冲液从光。 保持在冰上   每时每刻。 染色后保护载玻片免受光照。
      使用下表(或类似的表)来准备实验样品和阳性对照:

      组件

      总反应数
      总量
      平衡缓冲器
      45微升
      没有 o fslides + 1
      X微博
      核苷酸混合物
      5微升
      没有 o fslides + 1
      X微博
      TdT酶总量
      1微升
      没有 o fslides + 1
      X微博
      总计
      51微升
      没有 o fslides + 1
      X微博

      使用下表(或类似)来准备负控制:

      组件

      总反应数
      总量
      平衡缓冲器
      45微升
      1
      X微博
      核苷酸混合物
      5微升
      1
      X微博
      Milli-Q H sub 2 O 2/ 1微升
      1
      X微博
      总计
      51微升
      1
      X微博

    5. 使用镊子,取下塑料盖玻片,轻轻点击 滑动以除去多余的液体。 由于丢失的风险小心管理 部分。 用棉纸小心地擦干边缘 将幻灯片放在墙上的瓷砖上。
    6. 用微量移液管轻轻地放置50微升TdT培养缓冲液在载玻片上。
    7. 使用镊子,轻轻地放置一块塑料盖玻片在幻灯片的顶部,以均匀地分布液体
    8. 为了进行拖尾反应,将载玻片置于37℃的黑暗中 在实验室烘箱中60分钟。 倒置覆盖幻灯片 Coplin罐包含湿纸巾在底部,以确保高 湿度,并通过用铝箔覆盖保持在黑暗中
    9. 使用镊子,删除塑料盖玻片。 由于丢失部分的风险,请小心管理。
    10. 通过将幻灯片浸入Coplin来终止拖尾反应 瓶子含有2x SSC缓冲液。 在室温下孵育15分钟
    11. 通过将载玻片浸入含有1x PBS缓冲液的新鲜Coplin瓶中来洗涤载玻片。 在室温下孵育5分钟
    12. 重复两次步骤11使用一个新的Coplin罐,含有新鲜的1×PBS缓冲液 注意:准备在第14步中使用的0.5μg/ml PI。
    13. 让液体彻底排空,将幻灯片放在墙上的瓷砖上。
    14. 用0.5μg/ml PI覆盖每个切片。 解决方案必须完全 覆盖部分。 通常这可以用1ml溶液每次进行 滑动。
    15. 在室温下孵育5分钟。
    16. 洗 通过将载玻片转移到含有新鲜Coplin罐的组织 Milli-Q H 2 O并在室温下孵育5分钟。
    17. 重复步骤16两次。
      注意:准备在步骤19中使用的防褪色试剂。
    18. 让液体彻底排空,将幻灯片放在墙上的瓷砖上。
    19. 每个幻灯片添加150微升的抗褪色试剂,并盖住处理 包含具有玻璃盖片的部分的载片的部分。
    20. 用清晰的指甲油密封盖玻片的边缘,并允许 干燥至少5-10分钟。 样品可以立即检查或   在4℃下在黑暗中储存过夜,仅供下次观察 天。 我们通常在Leica TCS-SP2共焦显微镜中检查切片。   通过在自由处并入荧光素-dUTP产生荧光   使用激发来检测片段化DNA的3'-羟基末端 波长为488nm的TUNEL反应和561nm的PI,与 在492-550nm和581-625nm的范围内分别检测。

代表数据

用于猕猴桃组织中DNA断裂的TUNEL测定的这种改进方案依赖于通过用纤维素酶,Triton X-100和调整的蛋白酶K处理来改善组织的透化作用,用于产生的DNA酶I单位(1,500U/ml)增加的阳性对照,以及使用降低的IP浓度(0.5μg/ml)的复染的优化。这些修饰允许我们清楚地显示TUNEL的阴性和阳性细胞核之间的差异(图3A-C),与使用ApoAlert DNA片段化测定试剂盒的原始方案获得的结果相反,不是为植物组织特异设计的(图3D-F)。在图3A中,可观察到TUNEL阳性细胞核,因为它们显示亮绿色荧光,而在图3B中,所有存在的细胞核染成红色。最后,在图3C中,可以观察到所有存在的核,但是TUNEL阳性的那些染色为黄色,而那些TUNEL阴性的染色为浅红色或橙色。相反,使用ApoAlert 试剂盒(图3D-F),因为没有观察到TUNEL阳性细胞核(图3D),所有存在的细胞核可以以红色观察到(图3E)橙色复染色(图3F)。


图3.猕猴桃组织的阳性对照中TUNEL测定的比较。 A-C。使用本文描述的改进的TUNEL方案染色猕猴桃耻辱臂的阳性对照切片。 D-F。阳性对照 使用ApoAlert DNA片段化测定试剂盒(Clontech)染色猕猴桃耻辱臂的切片。 A,D。在488nm检测到的荧光用于观察TUNEL染色的细胞核(箭头)。 B,E。在561nm处检测荧光以观察IP染色的核(箭头)。 C,F。通过用TUNEL和IP(箭头)对染色的核的两个波长(488和561nm)的同时荧光检测进行计数染色。

食谱

  1. 固定溶液(100ml)
    乙醇:乙酸(3:1,v/v)洗脱 将75ml无水乙醇与25ml乙酸混合
  2. TBA溶液(100ml)
    TBA I

    纯TBA
    75 ml
    96%乙醇
    25 ml
    TBA II

    TBA I
    73.2 ml
    96%乙醇
    26.8 ml
    TBA III

    TBA II
    63.6毫升
    96%乙醇
    21.4 ml
    H sub 2 O
    15 ml
    TBA IV

    TBA III
    57 ml
    96%乙醇
    21.6 ml
    H sub 2 O
    21.4 ml
    TBA V

    TBA IV
    50 ml
    96%乙醇
    15 ml
    H sub 2 O
    35 ml
    TBA可在室温下固化
    如果发生这种情况,请在实验室烘箱中在38℃下加温
    在室温下储存在100 ml黑色玻璃瓶中,直到使用
  3. TBA:石蜡(3:1v/v)(100ml) 将25ml液体石蜡与75ml纯TBA混合 石蜡应在60℃下
    混合应使用液体
    在室温下储存在250 ml玻璃瓶中,直到使用
  4. 配方水(100ml)
    将3ml甲醛36.5-38%与97ml蒸馏水
    混合
  5. APTES涂层载玻片
    制备在丙酮(v/v)中的2%APTES 将玻片在室温下在2%APTES中浸泡3分钟
    将载玻片在丙酮中室温浸泡2分钟,
    重复两次
    将载玻片在蒸馏水中在室温下浸泡1分钟,
    允许风干(至少24小时)
    储存在室温下直到使用
  6. 1×PBS缓冲液(pH7.4,1L) 10x PBS缓冲液(pH7.4,1L)
    NaCl
    80克
    137 mM
    KCl
    2.2 g
    3 mM
    Na HPO 4
    14.4克
    10 mM
    KH 2 PO 4
    2.4克
    2 mM
    将试剂溶解在800ml Milli-Q H 2 O中 调整pH值
    将音量调整为1 L
    高压灭菌器
    合并100ml 10x PBS缓冲液和900ml Milli-Q H 2 O
  7. 4%甲醛的1×PBS缓冲液(pH7.4,1L)中 在通风橱下将40g多聚甲醛溶于500ml 1×PBS缓冲液中 在70℃下搅拌的同时加热溶液
    加入一滴或两滴硫酸以促进多聚甲醛稀释
    将音量调整为1 L
    储存在100 ml黑色瓶中,在4°C下最长1-2周,或在-20°C下无限期保存
  8. 2%纤维素酶Onozuka的1×PBS缓冲液(10ml)中 将0.2g纤维素酶溶解在6ml 1×PBS缓冲液中
    在4℃缓慢搅拌过夜孵育
    完成体积为10ml,过滤灭菌(0.2μm)
    储存于-20°C直到使用
  9. 0.5%Triton X-100的1x PBS缓冲液(10ml)中 将50μlTriton X-100溶于9.95 ml 1×PBS缓冲液中
    使用新鲜制备的溶液
  10. Tris-HCl(0.5M,pH8.0)(100ml) 将7.87g Tris-HCl溶于100ml蒸馏水中 用NaOH调节pH值
    高压灭菌并在室温下贮存
  11. EDTA(0.2M,pH8.0)(100ml) 将5.84g EDTA溶解在100ml蒸馏水中 用NaOH调节pH值
    高压灭菌并在室温下贮存
  12. 100mM Tris-HCl(pH8.0),50mM EDTA(3ml) 将2ml Tris-HCl缓冲液(0.5M,pH8.0)与1ml EDTA(0.2M,pH8.0)结合
  13. 蛋白酶K溶液(20μg/ml)(1ml) 将2μl的10mg/ml蛋白酶K与998μl的100mM Tris-HCl(pH8.0),50mM EDTA组合
  14. 1×DNase I缓冲液(100μl)
    用90μlMilli-Q H 2 O稀释10μl10x DNA酶I缓冲液
  15. 1,500 U/ml DNA酶I(75μl)
    合并45μlMilli-Q H Sub 2 O,22.5μl重组DNA酶I和7.5μl10×DNA酶I缓冲液
  16. PI溶液(0.5μg/ml,在PBS缓冲液中)(5ml) 库存:1mg/ml PI:将10mg PI溶解在10ml 1x PBS缓冲液中
    将原料在4℃下储存在黑暗中
    0.5μg/ml PI溶液:2.5μl原液和5ml 1x PBS缓冲液
  17. 抗褪色试剂(1ml)
    合并900μlPVA溶液(Citifluor固体填充试剂盒)和100μlAF100(Citifluor固体填充试剂盒)

致谢

这项研究由Xunta de Galicia(西班牙加利西亚地区政府;项目PGIDIT 04RAG291002PR)支持。我们感谢KiwiAtlánticoS.A.提供植物材料。我们感谢科罗拉多州的专家摄影协助,和B. Lueiro的技术援助。这是国际大学研究小组在木本植物的生物技术和生殖生物学(BioVitAc研究组,代码09IDI1705)的贡献。

参考文献

  1. ApoAlert DNA Fragmentation Assay Kit用户手册(Clontech,目录号:630107)
  2. Ferradas,Y.,Lopez,M.,Rey,M.and Gonzalez,M.V。(2014)。 猕猴桃耻辱臂中的程序性细胞死亡及其与有效传粉期和程序性阶段的关系。/a> Ann Bot 114(1):35-45
  3. 原位细胞死亡检测试剂盒,荧光素(Roche Diagnostics,目录号:11 684 795 910)
  4. Williams,J.H.,Jr.,Friedman,W.E.and Arnold,M.L。(1999)。 被子植物风格中的发育选择:使用配子DNA可视化种间花粉竞争。 Proc Natl Acad Sci USA 96(16):9201-9206。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ferradás, Y., López, M., Testillano, P., Rey, M. and González, M. V. (2015). TUNEL Assay in Kiwifruit Stigmatic Arms. Bio-protocol 5(7): e1434. DOI: 10.21769/BioProtoc.1434.
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