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Cytology and Microscopy: Immunolocalization of Covalently Modified Histone Marks on Barley Mitotic Chromosomes
细胞学和显微镜学:共价修饰组蛋白标记在大麦有丝分裂染色体上的免疫定位   

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Abstract

Barley is a diploid inbreeding crop with a genome of 5 GB organized into seven chromosomes. The relatively low chromosome number and their large size make barley an excellent model for chromosome cytogenetic studies of large genome cereal crops. Chromatin can be defined as euchromatin or heterochromatin. Euchromatin is gene-rich, less condensed, and transcriptionally active while the heterochromatin is gene-poor, remains highly condensed and has low transcriptional activity (Bartova et al., 2008; Sharakhov and Sharakhova, 2015). However, the mapping of nine Histone modifications has shown that this simple description is not accurate in barley. Instead, it has been shown that combinations of histones carrying different covalent modifications reveal 10 chromatin states partitioning barley chromosomes into three global environments (Baker et al., 2015). Briefly, in this protocol, barley roots (cv Morex) were collected, fixed in paraformaldehyde and squashed onto slides. Chromosome spreads were immunostained using antibodies against specific histone modifications, in particular H3K27me3, K3K27me1 and H3K9me2. We used confocal imaging to acquire stacked images and confirm the locations of these histone modifications on barley chromosomes.

Keywords: Barley, Chromosome, Immunocytology, H3K27me3, 3D micrcoscopy

Materials and Reagents

  1. SterilinTM 9 cm petri dish
(Thermo Fisher Scientific, catalog number: 1011RR )
  2. Whatman® 9 cm filter paper (Sigma-Aldrich, catalog number: Z240079 )
  3. Fine marker pen permanent (STABILO International GmbH)
  4. HB graphite pencil
  5. 15 ml centrifuge tube (any brand)
  6. Single edge razor blade (Thermo Fisher Scientific, catalog number: 11904325 )

  7. 1.5 ml microcentrifuge tubes
(VWR International, catalog number: 89000-028 )
  8. Microscope Polysine® slide (Thermo Fisher Scientific, Menzel Glaser, catalog number: 10143265 ) or Superfrost® plus micro slide (VWR International, catalog number: 48311-703 )
    Note: Currently, it is “Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 10143265 ”.
  9. Borosilicate glass coverslip, 25 x 50, thickness 0.13 mm (VWR International, catalog number: 631-0137 )
  10. Tissue paper
  11. PAP pen for immunostaining (Sigma-Aldrich, catalog number: Z377821 )
  12. 0.45 μm, 26 mm diameter syringe filter (Cole-Parmer, catalog number: UY-02915-58 )
  13. Glass Coplin staining jar (any brand)
  14. Seeds from barley cv. Morex
  15. Immersion oil, ImmersolTM W 2010 (Carl Zeiss)
  16. Autoclaved distilled water (Barnstead® E-pure 4 modules cartridges)
  17. 5% bleach (Domestos)
  18. 70% ethanol

  19. Liquid nitrogen
  20. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7030 )
  21. Antibodies
    1. Primary antibodies: anti-trimethyl-histone H3 (Lys27) antibody (H3K27me3) (Merck Millipore Corporation, catalog number: 07-449 ), anti-monomethyl-histone H3 (Lys27) antibody (K3K27me1) (Merck Millipore Corporation, catalog number: 07-448 ) and histone H3K9me2 antibody (pAb) (H3K9me2) (Active Motif, catalog number: 39375 )
      Note: Currently, it is “EMD Millipore Corporation, catalog numbers: 07-449 and 07-448 ”.
    2. Secondary antibodies: goat anti-Rabbit IgG (H+L), Alexa Fluor® 488 conjugate (Thermo Fisher Scientific, catalog number: A-11034 )
  22. Hoechst 33342 (10 mg/ml) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: H3570 )

  23. Vectashield antifade mounting medium (Vector Laboratories, catalog number: H-1000 )
  24. Clear nail varnish (Technic)
  25. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  26. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  27. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: 255793 )
  28. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5379 )
  29. Tween® 20 (Sigma-Aldrich, catalog number: P9416 )
  30. TritonTM X-100 (Sigma-Aldrich, catalog number: T8787 )
  31. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S8045 )
  32. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 ) 

  33. Cellulase Onozuka R10 (Duchefa Biochemie, catalog number: C8001 ) 

  34. Pectolyase Y23 (Dushefa Biochemie, catalog number: P8004 ) 

  35. 10x PBS (see Recipes)
  36. 1x PBS, 0.5% Triton® X-100 (see Recipes)
  37. 1 M NaOH (see Recipes)
  38. 4% formaldehyde (see Recipes)
  39. 0.1 M citric acid (see Recipes)
  40. 0.1 M sodium citrate (see Recipes)
  41. Citrate buffer (see Recipes)
  42. Enzyme mixture (see Recipes)
  43. Blocking solution (see Recipes)

Equipment

  1. Optoelectronically controlled small orbital shaker, 2,200 rpm, 230 V (IKA, model: VXR basic Vibrax® )
  2. Stereomicroscope (optional) (TEC Microscopes LTD., model: HM-4 )
  3. Light microscope (TEC Microscopes LTD., model: LM-2TR )
  4. Confocal microscope LSM 710 (Carl Zeiss)
  5. Objective (for Zeiss LSM 710): 60x APO C-apochromat 63x/1.20 W Korr M27
  6. DAPI fluorescence (blue 405, UV lamp) (Hoechst)
  7. Fitted 405 nm diode laser (for Hoechst acquisition on the LSM 710)
  8. Fitted 488 argon laser (for Alexa Fluor® 488 acquisition on the LSM 710)
  9. Moisture or wet chamber
    Note: Commercially available for immunohistochemistry application or home made by using a plastic box lined with wet paper and wax sheet or glass rod to keep the slides out of direct contact with water (Figure 1). The chamber needs to be closed so the humidity is preserved during the protocol.
  10. Aluminium slide tray, 20 slides capacity (Brunel Microscopes Ltd.)


    Figure 1. Wet chamber. The principle of the wet chamber is to keep the sample moist at all times.

Software

  1. Zen 2010 software (Carl Zeiss, version 6.0)

  2. Fiji (Image J, version 1.49m) (Schindelin et al., 2012) with deconvolution package (EPFL, Biomedical Imaging Group, Switzerland)
  3. Imaris (optional) (Bitplane AG, version 8.1.2)

Procedure

  1. Sample preparation
    Note: All steps were done at room temperature on the bench (no need for sterile conditions).
    1. Seeds sterilization
      1. Wash the seeds in 15 ml tube for 15 min in 5% bleach-shaking (5 ml). 

      2. Discard the bleach and rinse 3 times for 5 min in sterile water-shaking (5 ml) in the same tube. 

      3. Discard the water and wash the seeds in 70% ethanol for 5 min (5 ml) in the same tube. 

      4. Discard the Ethanol and rinse 3 times for 5 min in sterile water in the same tube.
    2. Seeds germination
      1. Place 4 to 6 seeds per petri dish containing a wet filter paper (sterile water). 

      2. Write the genotype and date on the lid with the fine marker pen. 

      3. Cover the plate with foil.
      4. Leave at room temperature for one day and let the seeds germinate for about 5 days at 4 °C (Figure 2).


        Figure 2. Root tips preparation. Barley seeds after 5 days germination (a) showing 3 to 4 roots. To prepare chromosomes spreads, only the yellow tips are taken (b, black lines), because they contain the dividing tissue while the remaining part of the roots is mainly composed of non-mitotic elongating cells. c. Close-up of the root tips under stereomicroscope.

  2. Slide preparation
    1. Chromosome spread preparation
      1. Cut the root tips (Figure 2a) under the stereomicroscope (Figure 2b) to isolate the yellow part (Figure 2c).
      2. Place in a 1.5 ml tube with 0.5 ml distilled water (dH2O).
      3. Place the tube on ice for at least 6 h (best overnight or 24 h), no shaking. Cells are arrested in metaphase during 6-24 h, therefore increasing the Mitotic Index Number of metaphase.
      4. Replace the cold water with fresh 4% formaldehyde (0.5 ml) and fix for 30 min at room temperature, no shaking. Formaldehyde has to be made fresh every time for reliability and using a fume hood (formaldehyde is carcinogenic).
      5. Rinse 3 x 5 min in 1x PBS (0.5 ml), in the fume hood. Discard in formaldehyde waste according to local laboratory rules.
      6. Digest roots with the enzyme mixture for 30-45 min at 37 °C. This step can be done in the 1.5 ml tube or any container such as an embryo dish. Root tips should become very soft after digestion. If not then, leave the root tips for further 20 min period(s) up to 2 h total digestion, until the roots can be easily squashed.
      7. Wash twice for 5 min with cold 1x PBS, 0.5% Triton X-100 at room temperature.
      8. Place 3 or 4 roots tips on a microscope slide (Figure 3a), add a 22 x 22 coverslip (see orientation on Figure 3b) and squash with your finger gently. Do not twist or roll the thumb from side to side. Be careful when handling the roots.
      9. Immerse the slide fully in liquid nitrogen using long forceps (wear appropriate safety protection according to the lab regulations). When the slide/coverslip is cooled enough (bubbling stops), remove the coverslip very quickly by inserting the razor blade (Figure 3b, grey shape) under one of the coverslip corners (Figure 3b).


        Figure 3. Squash method. For squashing, enzymatically digested root tips are placed between a microscope slide and a coverslip (a). The coverslip is placed as shown in (b) to be removed easily by using the edge of a razor blade (grey shape) under one side of the coverslip after freezing in liquid nitrogen.

      10. Lay the slide on an aluminium tray and let it air dry gently (see Note 1). Check the spread quality by looking at the chromosomes spread under a light microscope (using a 40x objective at least). Drying time normally takes no longer than 5-15 min at room temperature (21 °C), but this will depend on the temperature and humidity of the lab. It is best to process the slides as soon as they look dry.
        Note: Never use high temperatures as drying the slides too harshly will increase the background during immunostaining. Examine the samples quickly under the microscope. If necessary, add a drop of PBS onto the slide to check your spreads. For better results, use freshly made slides. Slides can be stored in the freezer for up to a month, but this will affect reproducibility. Leave the slide to dry for 5-15 min (depending on the lab environmental conditions). Add a drop of distilled water or PBS to the samples to keep them wet prior to adding the primary antibody.
      11. Use the slides immediately. 

        Note: Room temperature equals 21 °C, reproducibility of the protocol may vary if the temperature is higher than 21 °C, mainly by drying the sample too quickly.

  3. Immunostaining
    1. Use the PAP pen to circle the sample and let it dry.
      Note: It is possible to leave the slide for longer than 90 min, but longer exposure to the secondary antibody may increase the slide background with unbound antibody.
    2. Optional - Block the sample with the blocking solution for 20 min at room temperature in the wet chamber. This step did not make any difference for the antibodies used in this study but could improve the protocol for the use of other antibodies not yet tested.
    3. Apply 30 μl of primary antibody solution to the center of this circle. 

      Note: Primary antibody solution consists of 1 μl of histone antibody in 100 μl of blocking solution.
    4. Cover with a “soft” coverslip of Parafilm M® or cut autoclave bag. The dimension of these coverslips should match the slide dimensions (approximatively 22 x 50 mm).
    5. Place the slide in a wet chamber (see Figure1) for 30 min to 1 h at room temperature (see Note 4).
      Notes:
      1. The wet chamber comprises a plastic box lined with wet paper (Figure 1). The slide is placed on wax paper or glass rod, making sure it does not touch the wet surface.
      2. 10-15 min is normally enough to stain the cells, but this steps can be longer. However, this may increase unspecific background in the blue channel.
    6. Incubate slides overnight (and up to 48 h) in the fridge at 4 °C. Remove the slides from the fridge and leave them to warm up for at least 30 min at room temperature. 

    7. Wash the slides in 1x PBS, 0.5% Triton X-100 for at least 15 min (once 15 min or 3 x 5 min), in a glass Coplin staining jar, shaking at 100 rpm.
    8. Add 30 µl of the secondary antibody and repeat steps C3-4.
      1. Secondary antibody solution consisted of 1 μl of anti-rabbit Alexa Fluor® 488 in 600 μl of blocking solution. 

      2. Keep all steps in the dark from this point.
    9. Incubate 60-90 min at room temperature and repeat step C7. 

      Note: Slides mounted in Vectashield and sealed with nail varnish can be kept for months if stored in a dry place at 4 °C.
    10. Remove the excess buffer by keeping the slides vertically on a tissue paper. 

    11. Add 20 μl of 2 μg/ml Hoechst 33342 and incubate for 10-15 min at room temperature.
      Note: This step (step C11) can be skipped by using Vectashield already containing DNA staining. Other anti-fade solution could also be used although users should check the solution refracting index (1.45 for Vectashield) that can affect imaging.
    12. Remove the excess stain by placing the slide vertically on tissue paper for 5 sec and add one drop of Vectashield. 

    13. Carefully add a 22 x 50 coverslip, making sure that there are no bubbles underneath.
    14. Remove the excess of Vectashield by blotting the slide upside down on a tissue paper and seal with the nail varnish.
    15. Let it dry for at least 15 min and keep the slides in the dark and in the fridge until imaging.

  4. Imaging
    3D Confocal stack images (Figure 4a-c) were acquired with LSM-Zeiss 710 instrument and the ZEN software.


    Figure 4. Image analysis. Chromosomes Spread stained with Hoechst 33342 (a, d, g) and H3K27me3-Alexa488 (b, e, h). The images clearly show the distribution of H3K27me3 at the chromosomes ends (c). H3K27me3 modification (c) is mainly localized at one side of the nucleus (n) correlating with the so-called Rabl configuration (telomeres and centromeres are opposite side of the nucleus during Interphase). The sharpness of the raw images (a, b, c) can be improved by using deconvolution with Fiji (d, e, f) or the Imaris Gaussian filter (g, h, i). Scale bar, 5 µm.

    1. Locate your sample under the 10x objective using the DAPI fluorescence then switch to the C-Apochromat 63x/1.20 W Korr M27 objective [use ImmersolTM W (2010)]. 

    2. Locate your cell with DAPI and use the function Auto Exposure. 

    3. Zoom on your cell of choice and re-adjust the laser intensity. All images were taken with the following settings:
      1. Scan mode 0.5 μm stack section
      2. Pixel Dwell 2.27 μs
      3. Pinhole 67 μm
      4. Average line 2
      5. 12-bits
      6. Bidirectional
    4. Press run to acquire the image, name and save as .lsm files.

  5. Analysis
    Images were processed with Fiji (Schindelin et al., 2012) or Imaris 7.7.2 (Figure 3).
    1. Fiji
      1. In Fiji open your file using Plugins/Bio-Formats/Bio-Formats Importer 

      2. Unclick all option but “Split Channel” and “Autoscale”. 

      3. Select the image of the first channel and open the DeconvolutionLab Plugins (Vonesch and Unser, 2008). 

      4. Choose Tikhonov-Miller algorithm and choose between 2 to 5 iterations. Go to PSF and choose the image to deconvolve. Press run. Another image will be produced. Repeat the same for the second channel. 

      5. Go to Image/Merged Channel and choose your green and blue files names. Tick create composite and keep image, then click ok. You have now a merged 3D stacks. 

      6. Save using the Bio-Formats exporter and choose the .ome extension. This can be opened in many utility software later on. 

      7. Now select your 3D stack, go to Image/Stack/Z Project and choose maximum intensity. Change the colour by using Image/Colour/Channel Tool. Then choose Image/Type/RGB and save your images (Figure 4d-f) with the extension of your choice.
    2. IMARIS
      1. Open your image in Imaris and choose the option “Easy 3D”.
      2. Choose Image Processing/Smoothing/Gaussian filter for both channels-click OK.
      3. Click Snapshot, choose “Crop to fill the whole snapshot area”. 

      4. Select the dimension (either your dataset or 512 x 512) and choose the resolution (600 dpi for publication).
      5. Choose the file location and the file name and click “do Snapshot”. 

      6. Save your file in each channel by selecting/deselecting the channel in the Display Adjustment
panels (Figure 4g-i).

Representative data

Representative data are shown in Figure 4.

Recipes

  1. 10x PBS
    Dissolve the following in 800 ml dH2O 

    80 g of NaCl 

    2.0 g of KCl 

    14.4 g of Na2HPO4
    2.4 g of KH2PO4
    Adjust pH to 7.4

    Adjust volume to 1 L with additional dH2O

    Sterilized by autoclaving 

    Shelf life 6 months
    Dilute with dH2O to make 2x PBS or 1x PBS working solution (the shelf life of the diluted solutions is 1 month)
  2. 1x PBS, 0.5% TritonTM X-100
    Mix the following

    1 L of 1x PBS

    5 ml of Triton X-100

    Gently warm up the solution at 37 °C for 5 min to help dissolve Triton X-100
    Shelf life 1 month
  3. 1 M NaOH
    Dissolve 40 g of NaOH in 500 ml of dH2O
    Shelf life 6 months
  4. 4% formaldehyde
    For 25 ml of 4% formaldehyde solution (under a fume hood)
    Dissolve 1 g paraformaldehyde in 12.5 ml dH2O in a glass beaker

    Add one or two drop(s) of 1 M NaOH and seal the beaker with foil

    Heat at 60 °C at least 5-10 min, the solution must be transparent (do not boil) 

    Let it cool on ice until you can hold with your hand without burning
    Add 12.5 ml of 2x PBS
    The solution has to be fresh for best reproducibility
  5. 0.1 M citric acid
    Dissolve 2.1 g citric acid in 100 ml dH2O
    Autoclaved and stored in the fridge
    Shelf life 6 months (1 year if stored at 4 °C)
  6. 0.1 M sodium citrate
    Dissolve 2.94 g sodium citrate in 100 ml dH2O
    Autoclaved and stored in the fridge
    Shelf life 6 months (1 year if stored at 4 °C)
  7. Citrate buffer
    Make it freshly by mixing the following:
    444 μl of 0.1 M sodium citrate (2x)
    556 μl of citric acid (2x)
    Adjust to 10 ml with dH2O
    Best when freshly made but could also be stored in the fridge at 4 °C for 2 weeks
  8. Enzyme mixture
    Dissolve both enzymes in 10 ml citrate buffer:
    100 mg of Cellulase Onozuka R10

    100 mg of Pectolyase Y23 

    Aliquot out in 333 lots (optional: Filter through a 0.45 μm filter before) 

    Stored in freezer up to 1 year at -20 °C
    Thaw one aliquot and dilute with 667 μl citrate buffer to make 1 ml working solution
    Always made fresh and used on the same day
  9. Blocking solution
    Dissolve 0.15 g of BSA in 3 ml 1x PBS, 0.5% Triton X-100
    Always made fresh and used on the same day

Acknowledgments

We gratefully acknowledge the financial support from the grant BBSRC BB/I1022899/1 “The diversity and evolution of the gene component of the barley pericentromeric heterochromatin”, the European Community Grant FP7 MeioSys (222883) and the Scottish Government through RESAS work programme (WP5.2).

References

  1. Baker, K., Dhillon, T., Colas, I., Cook, N., Milne, I., Milne, L., Bayer, M. and Flavell, A. J. (2015). Chromatin state analysis of the barley epigenome reveals a higher-order structure defined by H3K27me1 and H3K27me3 abundance. Plant J 84(1): 111-124.
  2. Bartova, E., Krejci, J., Harnicarova, A., Galiova, G. and Kozubek, S. (2008). Histone modifications and nuclear architecture: a review. J Histochem Cytochem 56(8): 711-72
  3. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J. Y., White, D. J., Hartenstein, V., Eliceiri, K., Tomancak, P. and Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7): 676-682.
  4. Sharakhov, I. V. and Sharakhova, M. V. (2015). Heterochromatin, histone modifications, and nuclear architecture in disease vectors. Curr Opin Insect Sci 10: 110-117.
  5. Vonesch, C. and Unser, M. (2008). A fast thresholded landweber algorithm for wavelet-regularized multidimensional deconvolution. IEEE Trans Image Process 17(4): 539-549.

简介

大麦是一种二倍体近亲繁殖作物,基因组为5 GB,组织成7条染色体。相对低的染色体数量及其大小使大麦成为大型基因组谷物作物的染色体细胞遗传学研究的优良模型。染色质可以定义为真核细胞色素或异染色质。现代染色质是富含基因的,较少缩合的和转录活性的,而异染色质是基因缺乏的,保持高度缩合并具有低转录活性(Bartova等人。 Sharakhov和Sharakhova,2015)。然而,九个组蛋白修饰的映射显示,这种简单描述在大麦中不准确。相反,已经显示携带不同共价修饰的组蛋白的组合揭示了将大麦染色体分成三个全球环境的10个染色质状态(Baker等人,2015)。简言之,在该方案中,收集大麦根(cv Morex),在多聚甲醛中固定并压在载玻片上。使用针对特定组蛋白修饰的抗体,特别是H3K27me3,K3K27me1和H3K9me2对染色体涂片进行免疫染色。我们使用共聚焦成像获得堆叠图像,并确认这些组蛋白修饰在大麦染色体的位置。

关键字

材料和试剂

  1. Sterilin TM 9cm培养皿(Thermo Fisher Scientific,目录号:1011RR)
  2. Whatman 9cm滤纸(Sigma-Aldrich,目录号:Z240079)
  3. Fine marker pen permanent(STABILO International GmbH)
  4. HB石墨铅笔
  5. 15ml离心管(任何品牌)
  6. 单刃剃刀刀片(Thermo Fisher Scientific,目录号:11904325)
  7. 1.5ml微量离心管(VWR International,目录号:89000-028)
  8. 显微镜Polysine 载玻片(Thermo Fisher Scientific,Menzel Glaser,目录号:10143265)或Superfrost plus微载玻片(VWR International,目录号:48311-703)
    注意:目前,"Thermo Fisher Scientific,Thermo Scientific TM ,目录号:10143265" br />
  9. 硼硅酸盐玻璃盖玻片,25×50,厚度0.13mm(VWR International,目录号:631-0137)
  10. 纸巾
  11. 用于免疫染色的PAP笔(Sigma-Aldrich,目录号:Z377821)
  12. 0.45μm,26mm直径的注射器过滤器(Cole-Parmer,目录号:UY-02915-58)
  13. 玻璃Coplin染色罐(任何品牌)
  14. 种子从大麦cv。 莫雷克斯
  15. 浸没油,Immersol TM W 2010(Carl Zeiss)
  16. 高压蒸馏水(Barnstead E-pure 4模块盒)
  17. 5%漂白剂(Domestos)
  18. 70%乙醇
  19. 液氮
  20. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A7030)
  21. 抗体
    1. 一级抗体:抗三甲基组蛋白H3(Lys27)抗体(H3K27me3)(Merck Millipore Corporation,目录号:07-449),抗单甲基组蛋白H3(Lys27)抗体(K3K27me1)(Merck Millipore Corporation, 07-448)和组蛋白H3K9me2抗体(pAb)(H3K9me2)(Active Motif,目录号:39375)
      注意:目前,它是"默克密理博公司,目录号:07-449和07-448"。
    2. 二抗:山羊抗兔IgG(H + L),Alexa Fluor 488缀合物(Thermo Fisher Scientific,目录号:A-11034)
  22. Hoechst 33342(10mg/ml)(Thermo Fisher Scientific,Molecular Probes ,目录号:H3570)
  23. Vectashield防褪色封固剂(Vector Laboratories,目录号:H-1000)
  24. 清除指甲油(Technic)
  25. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  26. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  27. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:255793)
  28. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P5379)
  29. Tween 20(Sigma-Aldrich,目录号:P9416)
  30. TritonX-100(Sigma-Aldrich,目录号:T8787)
  31. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:S8045)
  32. 多聚甲醛(Sigma-Aldrich,目录号:P6148)
  33. 纤维素酶onozuka R10(Duchefa Biochemie,目录号:C8001)
  34. Pectolyase Y23(Dushefa Biochemie,目录号:P8004)
  35. 10x PBS(请参阅配方)
  36. 1x PBS,0.5%Triton X-100(参见配方)
  37. 1 M NaOH(见配方)
  38. 4%甲醛(见配方)
  39. 0.1 M柠檬酸(见配方)
  40. 0.1 M柠檬酸钠(见配方)
  41. 柠檬酸缓冲液(见配方)
  42. 酶混合物(见配方)
  43. 阻塞溶液(参见配方)

设备

  1. 光电控制的小轨道摇床,2200rpm,230V(IKA,型号:VXR basic Vibrax )
  2. 立体显微镜(可选)(TEC显微镜有限公司,型号:HM-4)
  3. 光学显微镜(TEC显微镜有限公司,型号:LM-2TR)
  4. 共聚焦显微镜LSM 710(Carl Zeiss)
  5. 目标(对于Zeiss LSM 710):60x APO C-apochromat 63x/1.20W Korr M27
  6. DAPI荧光(蓝色405,UV灯)(Hoechst)
  7. 安装的405 nm二极管激光器(用于LSM 710上的Hoeschst采集)
  8. 配备488氩激光器(用于LSM 710上的Alexa Fluor ® 488采集)
  9. 潮湿或潮湿的房间
    注:市售可用于免疫组织化学应用或通过使用衬有湿纸和蜡片或玻璃棒的塑料盒来保持载片不与水直接接触(图1)。 室需要关闭,以便在协议期间保持湿度。
  10. 铝滑动托盘,20张幻灯片容量(Brunel Microscopes Ltd.)


    图1.湿室。 湿室的原理是保持样品始终保持湿润。

软件

  1. Zen 2010软件(Carl Zeiss,版本6.0)
  2. 斐济(Image J,版本1.49m)(Schindelin等人,2012年)与去卷积包(EPFL,Biomedical Imaging Group,Switzerland)
  3. Imaris(可选)(Bitplane AG,版本8.1.2

程序

  1. 样品准备
    注意:所有步骤都在室温下在工作台上进行(不需要无菌条件)。
    1. 种子灭菌
      1. 洗涤种子在15毫升管中15分钟,在5%漂白摇动(5毫升)。
      2. 弃去漂白剂,并在同一管中的无菌水摇动(5ml)中漂洗3次,每次5分钟。
      3. 弃去水并在同一管中在70%乙醇中洗涤种子5分钟(5ml)。
      4. 丢弃乙醇,并在同一管中的无菌水中漂洗3次,每次5分钟。
    2. 种子发芽
      1. 每个培养皿放置4到6个种子,含有湿滤纸(无菌水)。
      2. 使用细符号笔在盖子上书写基因型和日期。
      3. 用铝箔覆盖板。
      4. 在室温下放置一天,让种子在4℃下发芽约5天(图2)。


        图2.根尖制备。 5天后的大麦种子萌发(a)显示3到4个根。为了制备染色体扩散,仅取黄色尖端(b,黑色线),因为它们含有分裂组织,而根的剩余部分主要由无有丝分裂的延伸细胞组成。 C。在立体显微镜下的根尖的特写
  2. 幻灯片准备
    1. 染色体扩散制剂
      1. 在立体显微镜下切下根尖(图2a)(图2b),分离黄色部分(图2c)。
      2. 置于具有0.5ml蒸馏水(dH 2 O)的1.5ml管中
      3. 将管置于冰上至少6小时(最好过夜或24小时),不摇动。 细胞在6-24小时期间在中期停止,因此增加中期的有丝分裂指数
      4. 用新鲜的4%甲醛(0.5ml)替换冷水,在室温下固定30分钟,无振荡。 甲醛必须每次使用新鲜的可靠性和使用通风橱(甲醛致癌)
      5. 在通风橱中,在1x PBS(0.5 ml)中冲洗3 x 5分钟。根据当地实验室规则丢弃甲醛废物。
      6. 用酶混合物在37℃消化根部30-45分钟。该步骤可以在1.5ml管或任何容器例如胚盘中进行。根尖应消化后变得非常柔软。如果不是,然后留下根提示进一步20分钟的时间,总共2小时消化,直到根可以很容易挤压。
      7. 用冷的1x PBS,0.5%Triton X-100在室温下洗涤两次,每次5分钟
      8. 将3或4根根尖放在显微镜载玻片上(图3a),添加一个22 x 22盖玻片(见图3b上的方向),用你的手指轻轻地挤压。不要从一侧到另一侧扭转或滚动拇指。处理根时要小心。
      9. 使用长镊子将载玻片完全浸入液氮中(根据实验室规定佩戴合适的安全防护装置)。当载玻片/盖玻片充分冷却(起泡停止)时,通过将剃刀刀片(图3b,灰色形状)插入盖玻片角下(图3b),非常快速地除去盖玻片。


        图3. Squash方法。对于挤压,酶促消化的根尖置于显微镜载玻片和盖玻片(a)之间。如在(b)中所示放置盖玻片,以便在液氮中冷冻后,使用剃刀刀片的边缘(灰色形状)在盖玻片的一侧下容易地除去。
      10. 将载玻片放在铝托盘上,让其轻轻空气干燥(见注1)。通过观察在光学显微镜下(至少使用40x物镜)扩散的染色体检查扩散质量。干燥时间通常在室温(21℃)下不超过5-15分钟,但这将取决于实验室的温度和湿度。最好是一旦看到干燥处理幻灯片。
        注意:切勿使用高温,因为干燥载玻片太苛刻会增加免疫染色时的背景。在显微镜下迅速检查样品。如有必要,请在幻灯片上添加一滴PBS以检查点差。为了获得更好的效果,请使用新制作的幻灯片。幻灯片可以存储在冰箱中长达一个月,但这将影响重现性。让载玻片干燥5-15分钟(取决于实验室环境条件)。在加入一抗之前,向样品中加入一滴蒸馏水或PBS以保持其湿润。
      11. 立即使用幻灯片。
        注意:室温等于21°C,如果温度高于21°C,协议的重现性可能会有所不同,主要是因为样品干燥过快。

  3. 免疫染色
    1. 使用PAP笔圈起样品,让其干燥。
      注意:可以将载玻片离开超过90分钟,但是更长时间暴露于二级抗体可以增加具有未结合抗体的载玻片背景。
    2. 可选 - 在室温下在封闭溶液中封闭样品20分钟。 此步骤对本研究中使用的抗体没有任何区别,但可以改善使用尚未测试的其他抗体的方案。
    3. 应用30微升一抗溶液到这个圆的中心。
      注意:一级抗体溶液由100μl阻断溶液中的1μl组蛋白抗体 。
    4. 用Parafilm M 或切割的高压釜袋的"软"盖玻片覆盖。 这些盖玻片的尺寸应与幻灯片尺寸(近似为22 x 50 mm)相匹配。
    5. 将载玻片置于湿室中(见图1)在室温下30分钟至1小时(见注4)。
      注意:
      1. 湿室包括衬有湿纸的塑料盒(图1)。 将载玻片置于蜡纸或玻璃棒上,确保其不接触湿表面。
      2. 10-15分钟通常足以染色细胞,但这个步骤可以更长。 但是,这可能会增加蓝色通道中的非特定背景。
    6. 孵育玻璃过夜(和长达48小时)在冰箱在4℃。 从冰箱中取出载玻片,让它们在室温下预热至少30分钟。
    7. 在玻璃Coplin染色罐中以100rpm振荡,在1x PBS,0.5%Triton X-100中洗涤载玻片至少15分钟(一次15分钟或3×5分钟)。
    8. 加入30μl的第二抗体,并重复步骤C3-4。
      1. 第二抗体溶液由在600μl封闭溶液中的1μl抗兔Alexa Fluor 488组成。
      2. 从这一点保持所有步骤在黑暗中。
    9. 在室温下孵育60-90分钟,并重复步骤C7。
      注意:安装在vectashield和用指甲油密封的幻灯片可以保存几个月,如果存储在4℃的干燥地方。
    10. 通过将载玻片垂直保持在薄纸上来移除多余的缓冲液。
    11. 加入20微升2微克/毫升Hoechst 33342,并在室温下孵育10-15分钟 注意:可以通过使用已经包含DNA染色的vectashield来跳过该步骤(步骤C11)。也可以使用其他防褪色解决方案,虽然用户应该检查影响成像的解折射指数(1.45对于vectashield)。
    12. 通过将载玻片垂直放置在薄纸上5秒钟,并加入一滴Vectashield去除多余的污渍。
    13. 小心地添加一个22 x 50的盖玻片,确保下面没有气泡
    14. 通过将载玻片上下颠倒在薄纸上并用指甲油密封,除去多余的Vectashield。
    15. 让它干燥至少15分钟,并保持幻灯片在黑暗中和冰箱里,直到成像
  4. 成像
    使用LSM-Zeiss 710仪器和ZEN软件采集3D共聚焦堆叠图像(图4a-c)。


    图4.图像分析。染色体用Hoechst 33342(a,d,g)和H3K27me3-Alexa488(b,e,h)染色的扩散染色。图像清楚地显示H3K27me3在染色体末端的分布(c)。 H3K27me3修饰(c)主要定位在与所谓的Rab1构型相关的核(n)的一侧(端粒和着丝粒在间期期间核的相对侧)。可以通过使用具有Fiji(d,e,f)或Imaris高斯滤波器(g,h,i)的反卷积来提高原始图像(a,b,c)的锐度。比例尺,5μm。

    1. 使用DAPI荧光定位您的样品在10x物镜下,然后切换到C-Apochromat 63x/1.20W Korr M27物镜[使用Immersol TM sup/W(2010)]。
    2. 使用DAPI定位您的单元格,并使用自动曝光功能。
    3. 放大您选择的单元格并重新调整激光强度。 所有图像采用以下设置:
      1. 扫描模式0.5μm堆叠部分
      2. 像素驻留2.27μs
      3. 针孔67μm
      4. 平均第2行
      5. 12位
      6. 双向
    4. 按运行以获取图像,命名并另存为.lsm文件
  5. 分析
    图像用Fiji(Schindelin等人,2012)或Imaris 7.7.2(图3)处理。
    1. 斐济
      1. 在斐济打开您的文件使用插件/生物格式/生物格式导入器
      2. 取消选中所有选项,但"拆分频道"和"自动缩放"。
      3. 选择第一个通道的图像,并打开DeconvolutionLab插件(Vonesch和Unser,2008)。
      4. 选择Tikhonov-Miller算法,并选择2到5次迭代。 转到PSF并选择要解卷积的图像。 按运行。 将生成另一个图像。 对第二个通道重复同样的操作。
      5. 转到图像/合并频道,然后选择您的绿色和蓝色文件名称。 勾选创建复合并保持图像,然后单击确定。 你有现在一个合并的3D堆栈。
      6. 使用Bio-Formats导出器保存并选择.ome扩展名。 这可以在许多实用软件中打开。
      7. 现在选择你的3D堆栈,去图像/堆栈/Z项目并选择最大强度。 使用图像/颜色/频道工具更改颜色。 然后选择图像/类型/RGB,并使用您选择的扩展名保存图像(图4d-f)。
    2. IMARIS
      1. 在Imaris中打开图片,然后选择"简易3D"选项。
      2. 为两个通道选择图像处理/平滑/高斯滤镜 - 单击确定。
      3. 单击快照,选择"裁剪以填充整个快照区域"。
      4. 选择维度(数据集或512 x 512),然后选择分辨率(600 dpi用于发布)。
      5. 选择文件位置和文件名,然后单击"执行快照"。
      6. 通过在显示调整面板中选择/取消选择通道,将文件保存在每个通道中(图4g-i)。

代表数据

代表性数据显示在图4中。

食谱

  1. 10x PBS
    将以下物质溶解在800ml dH 2 O中 80克NaCl
    2.0克KCl
    14.4g的Na 2 HPO 4
    2.4g的KH 2 PO 4 sub/
    将pH调节至7.4
    使用附加的dH <2> O调整音量至1 L
    高压灭菌
    灭菌保质期6个月
    用dH 2 O稀释以制备2x PBS或1x PBS工作溶液(稀释溶液的保存期为1个月)
  2. 1x PBS,0.5%Triton TM sup-X-100 混合以下
    1 L的1x PBS
    5ml Triton X-100 轻轻加热溶液在37°C 5分钟,以帮助溶解Triton X-100
    保质期1个月
  3. 1 M NaOH
    将40g NaOH溶解在500ml dH 2 O中 保质期6个月
  4. 4%甲醛
    对于25ml的4%甲醛溶液(在通风橱下)
    在玻璃烧杯中将1g多聚甲醛溶解在12.5ml dH 2 O中。
    加入一滴或两滴1M NaOH,并用箔片
    密封烧杯 在60℃下加热至少5-10分钟,溶液必须是透明的(不沸腾)
    让它在冰上冷却,直到你能握住你的手,而不燃烧
    加入12.5ml的2x PBS
    该溶液必须是新鲜的,以获得最佳的再现性
  5. 0.1M柠檬酸
    将2.1g柠檬酸溶于100ml dH 2 O中 高压灭菌并储存在冰箱中
    保质期6个月(1年,如果储存在4℃)
  6. 0.1M柠檬酸钠 将2.94g柠檬酸钠溶于100ml dH 2 O中 高压灭菌并储存在冰箱中
    保质期6个月(如果储存在4℃下1年)
  7. 柠檬酸盐缓冲液
    混合以下内容,使其新鲜:
    444μl的0.1M柠檬酸钠(2x)
    556μl柠檬酸(2x)
    用dH 2 O调节至10ml。
    最好当新鲜制作,但也可以存储在冰箱在4°C 2周
  8. 酶混合物
    将两种酶溶解在10ml柠檬酸盐缓冲液中:
    100mg纤维素酶Onozuka R10
    100mg的果胶酶Y23
    在333个批次(可选:过滤通过0.45μm过滤器)
    储存在-20°C的冰柜中1年,
    解冻一个等分试样,用667μl柠檬酸盐缓冲液稀释,得到1ml工作溶液 始终保持清新并在同一天使用
  9. 封锁解决方案
    将0.15g BSA溶于3ml 1×PBS,0.5%Triton X-100中 始终保持清新并在同一天使用

致谢

我们衷心感谢授予BBSRC BB/I1022899/1"大麦分生孢子核异染色质的基因组分的多样性和进化",欧洲共同体授权FP7 MeioSys(222883)和苏格兰政府通过RESAS工作程序(WP5 .2)。

参考文献

  1. Baker,K.,Dhillon,T.,Colas,I.,Cook,N.,Milne,I.,Milne,L.,Bayer,M.and Flavell,AJ(2015)。  大麦表观基因组的染色质状态分析揭示了由H3K27me1和H3K27me3丰度定义的高阶结构。 Plant J 84(1):111-124。
  2. Bartova,E.,Krejci,J.,Harnicarova,A.,Galiova,G。和Kozubek,S。(2008)。  组蛋白修饰和核结构:综述。 Histochem Cytochem 56(8):711-72
  3. Schindelin,J.,Arganda-Carreras,I.,Frize,E.,Kaynig,V.,Longair,M.,Pietzsch,T.,Preibisch,S.,Rueden,C.,Saalfeld,S.,Schmid,B这样的话,我们可以通过使用一个简单的方法来解决这个问题,这个问题的解决方案是: /www.ncbi.nlm.nih.gov/pubmed/22743772"target ="_ blank">斐济:用于生物图像分析的开源平台国家方法 9(7) ):676-682。
  4. Sharakhov,IV和Sharakhova,MV(2015)。  异染色质,组蛋白修饰和核结构在疾病载体中。 Curr Opin Insect Sci 10:110-117。
  5. Vonesch,C。和Unser,M。(2008)。  用于小波正则化多维解卷积的快速阈值Landweber算法。 IEEE Trans Image Process 17(4):539-549。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Colas, I., Baker, K. and Flavell, A. J. (2016). Cytology and Microscopy: Immunolocalization of Covalently Modified Histone Marks on Barley Mitotic Chromosomes. Bio-protocol 6(12): e1841. DOI: 10.21769/BioProtoc.1841.
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