欢迎您, 登录 | 注册

首页 | English

X
加载中

The protocol describes preparation of young developing maize kernels for microscopical analysis of nuclei in tissue sections. The fixative FAA (formaldehyde, acetic acid and ethanol) is suitable for preservation of nuclear morphology and allows for quantitative staining of DNA with Schiff reagent in Feulgen staining. The fixation and embedding protocol may be used also for various other histology staining procedures, but care must be taken as the cytoplasm usually shrinks a bit using this procedure. The protocol was used for analysis of seed development of various maize lines, mutants and maize relatives (Vilhar et al., 2002; Kladnik et al., 2006; Dermastia et al., 2009; Bernardi et al., 2012).

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

Maize Kernels – Fixation in FAA, Embedding, Sectioning and Feulgen Staining
玉米籽粒的固定(FAA)、包埋、切片及染色

植物科学 > 植物发育生物学 > 形态建成
作者: Aleš Kladnik
Aleš KladnikAffiliation: Department of Biology, University of Ljubljana, Biotechnical Faculty, Ljubljana, Slovenia
For correspondence: ales.kladnik@bf.uni-lj.si
Bio-protocol author page: a412
Vol 3, Iss 15, 8/5/2013, 5206 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.835

[Abstract] The protocol describes preparation of young developing maize kernels for microscopical analysis of nuclei in tissue sections. The fixative FAA (formaldehyde, acetic acid and ethanol) is suitable for preservation of nuclear morphology and allows for quantitative staining of DNA with Schiff reagent in Feulgen staining. The fixation and embedding protocol may be used also for various other histology staining procedures, but care must be taken as the cytoplasm usually shrinks a bit using this procedure. The protocol was used for analysis of seed development of various maize lines, mutants and maize relatives (Vilhar et al., 2002; Kladnik et al., 2006; Dermastia et al., 2009; Bernardi et al., 2012).
Keywords: plant biology(植物生物学), histology(组织学), fixation(固定), DNA staining(DNA染色), seed(种子)

[Abstract]

Materials and Reagents

  1. Maize Kernels
    At different developmental stages; kernels up to 20 days after pollination (DAP) usually fix and section well, at later stages the endosperm becomes progressively dry and hard, causing the sections to crumble and tear while sectioning on the microtome
  2. Paraplast Plus (Pelco, catalog number: 18393 ; Sherwood Medical, catalog number: 8889-502005; or Sigma-Aldrich, catalog number: P3683 )
  3. Glacial acetic acid (Sigma-Aldrich, catalog number: A6283 )
  4. Formaldehyde solution (histological grade, 37 wt.% in H2O) (Sigma-Aldrich, catalog number: 533998 )
  5. Ethanol (96% and absolute)
  6. Tert-butanol (TBA) (Sigma-Aldrich, catalog number: B85927 )
  7. Pararosaniline hydrochloride (Sigma-Aldrich, catalog number: P3750 )
  8. Potassium metabisulfite (K2S2O5) (Sigma-Aldrich, catalog number: 31268 )
  9. Xylene (Xylenes, histological grade) (Sigma-Aldrich, catalog number: 534056 )
  10. 5 M HCl (Merck-Millipore, catalog number: 1099110001 )
  11. Decolorizing charcoal (BDH, GB) (Sigma-Aldrich, catalog number: 161551 )
  12. DPX mounting medium (Sigma-Aldrich, catalog number: 0 6522 )
  13. Formaldehyde – acetic acid – ethanol (FAA) (see Recipes)
  14. Dehydrating solutions (see Recipes)
  15. Schiff reagent (see Recipes)

Equipment

  1. Glass vials (volume ~20 ml)
  2. Glass fiber filters (Microfibre filters, Whatman GF/C)
  3. Scalpels, razor blades
  4. Metal forceps (use dedicated forceps for wax work, they are difficult to clean)
  5. Disposable plastic Pasteur pipettes
  6. Vacuum desiccator (Bel-Art, model: 420100000 )
  7. Staining dishes (for melting Paraplast Plus) (Electron Microscopy Sciences, catalog number: 70312-21 )
  8. Hot plate (set on low – around 68 °C), with aluminum foil tent to keep the heat inside
  9. Alcohol burner
  10. Plastic or metal molds
    Disposable Base Mold (Pelco, catalog number: 27147 )
    Tissue-Tek Base Mold (Sakura, catalog number: 4123 )
  11. Tissue embedding cassette bases (Pelco, catalog number: 27168-1 ) or embedding rings (Pelco, catalog number: 27169-1 ) or Tissue-Tek Embedding Rings (Sakura, catalog number: 4151 )
  12. Small beaker with molten Paraplast Plus (dedicated for wax work)
  13. Tray with a thin layer of cold water (cool it by adding ice)
  14. Ice bucket filled with ice
  15. Flat ice packet (frozen cooling pack) or brass plate (optional)
  16. Rotary microtome
  17. Disposable microtome blades
  18. Small brush
  19. Forceps with sharp ends
  20. Single-edged razorblade
  21. Dark cardboard plate
  22. Stereomicroscope
  23. Coplin jars (Electron Microscopy Sciences, catalog number: 70316-02 )

Procedure

  1. Fixation in FAA
    1. Aliquot FAA fixative in glass vials (~20 ml vials, 10 ml fixative). Keep vials on ice, fixative should be cold when you put tissue in. Cut tissue with a scalpel or razor blade into pieces, that have at least one dimension smaller than 2 or 3 mm, and immediately immerse in cold fixative.
      Note: When cutting the tissue take into consideration the type of sections you wish to obtain.
    2. Place open vials with fixed material in the vacuum desiccator and expose them to moderate vacuum for 15 min to pull the air out of the tissue. If the tissue doesn't sink when you release vacuum, apply and release the vacuum one more time. After vacuuming, replace fixative in vials with fresh fixative.
    3. Fix overnight at 4 °C (refrigerator).

  2. Dehydration in a series of TBA
    1. Keep pure TBA (1 L) in a warm place before use (e.g. on the top of an incubation oven), it freezes below 25 °C. Prepare dehydration solutions and store solutions a – e on room temperature (RT) and f – h on the incubating oven.
    2. Dehydrate tissue in each step for an hour to one day, depending on tissue size (6 DAP maize kernels for half a day, 12 DAP maize kernels for one day each step). Steps with dehydration solution a – e should be on RT, steps f – h in the incubation oven at 56-60 °C.
      Use a plastic Pasteur pipette to pull the old solution from the vial, then replace with equal amount of next solution (you need a relatively much larger volume of dehydrating solution compared to your sample). Mark dehydration progress on the label on vial. Alternatively, you can pour the old solution out of the vial and replace with a new one (you don't need to completely drain the vial). Work in a fume hood. In the last change of TBA, fill the vial only to the half of the vial volume, so that you will leave enough room for the adding of the Paraplast Plus in the next step.

  3. Embedding in paraffin (Paraplast Plus)
    1. Keep molten Paraplast Plus and plastic pipettes in the incubation oven at 56-60 °C. You need a large stock of molten paraffin, because it takes a long time to melt (e.g. two rectangular histology staining dishes holding ~300 ml).
    2. Add pellets of Paraplast Plus wax in vials half filled with TBA. Alternatively, you can add equal volume of molten wax to the TBA in the vials. Paraplast Plus has a fine structure and additives (DMSO), which help in better penetration into the tissue, which is very important for trouble-free sectioning. Keep in 56-60 °C oven, with the vial cap attached and mix a few times, when the wax is completely melted. Leave in the oven overnight.
    3. The next day, pour or pipette away the TBA/wax mixture in a waste container in a fume hood (you can make a small waste container out of aluminum foil). Add fresh molten wax, and leave in oven overnight with vials open, so that all the TBA evaporates. The next day change the wax with fresh one several times (use a warm transfer pipette or pour away), for example twice a day for two days.
  4. Casting wax blocks
    1. Set air condition in the lab on warmer (if it is too cold, the wax will solidify too quickly).
    2. Equipment:
      1. Hot plate (set on low – around 68 °C), with aluminum foil tent to keep the heat inside
      2. Metal forceps (use dedicated forceps for wax work, they are difficult to clean)
      3. Alcohol burner
      4. Plastic or metal molds
      5. Embedding rings or embedding cassete bases
      6. A small beaker with molten Paraplast Plus (also has to be dedicated for wax work)
      7. A tray with a thin layer of cold water (cool it by adding ice)
      8. An ice buckets filled with ice
      9. Flat ice packet (frozen cooling pack) or brass plate (optional)
    3. Keep a small amount of molten wax in a beaker in the aluminium foil tent in the hot plate. We also protect the hot plate with a layer of aluminium foil to keep it clean from wax. If the plate is not precisely thermo-regulated check the plate temperature all the time – if it gets too hot to touch, you have to turn it off and then turn it on when the wax starts to solidify. Prolonged temperatures over 62 °C will damage Paraplast Plus. Also, if using metal molds, keep them on a hot plate before using them.
    4. Take one vial with samples out of the oven at a time, and place it on the hot plate. Label the same number of embedding rings as the number of samples with a pencil or waterproof marker. Pour some wax in the mold, take one sample out of the vial with forceps warmed in a flame of alcohol burner.
      Note: Be careful, just pass forceps through the flame for a short time, if smoke is coming off the forceps, they are too hot.
      Orient the sample in the mold on the hotplate considering the type of sections you wish to obtain later. Carefully transfer the mold on the brass-plate/ice-packet or just on the table, so that the bottom layer of wax solidifies and fixes the sample in place. If the sample is difficult to position on the hot plate, you can position it now, when the bottom layer of wax is solid. Fill the mold with molten wax to the edge, and cover with the labeled embedding ring. Then transfer the mold into the tray with cold water and ice and leave it there for about 10 min so that wax solidifies completely. Then put the mold on the ice, wait for a few minutes and then remove the wax block from the mold (the cooling with ice helps the block getting loose from the mold). Put the mold back on a hotplate to re-use it. We usually process 5 to 10 blocks at a time. Store the blocks in the refrigerator at 4 °C (see Figure 1).


      Figure 1. Schematic drawing of workplace organization for embedding tissue samples in paraffin.

  5. Sectioning
    1. Equipment:
      1. Rotary microtome
      2. Disposable microtome blades
      3. A small brush
      4. A forceps with sharp ends
      5. A single-edged razorblade
      6. Dark cardboard plate (for putting the sections on and observing them)
      7. A stereomicroscope (optional but recommended)
      8. Hot plate set on 40 °C
      9. A small jar with distilled water
      10. A plastic pipette
      11. Cleaned objective microscope slides, frosted on one edge
      12. A pencil
    2. Trim the wax block around the sample with a single-edged razor blade so that the upper and lower edges are parallel, and the left and right edges are at an angle, so that the lower edge of the block is longer than the upper. Also the sides of the block should not be vertical but inclined – always cut the block with the razor blade away from the sample in clean cuts from the top to the bottom of the block (see Figure 2).


      Figure 2. Sectioning paraffin-embedded tissue samples. Top: Directions of trimming the paraffin around the sample. Bottom: sectioning on the microtome and arranging the sections on a slide.

    3. Fix the embedding ring with the sample in the sample holder on the microtome. Set the thickness of sections (usually 10 to 20 μm) and the cutting angle (around 7 degrees). Start sectioning; the first few sections won't be good, so remove them with a small brush, always away from the blade, not towards it! When the surface of the sample evens, the ribbon will start forming. Cut enough sections to fit them on a dark cardboard, and cut the ribbon with a razor blade. Cut several ribbons, until you are sure that you have cut the region of interest, and then look at the sections with a stereomicroscope, to locate the interesting sections (e.g. median longitudinal sections of the kernel).
    4. Label the objective slides with a pencil, put them on a hot plate (at 40 °C) and apply some water on them (to almost cover them). Cut the ribbon in smaller regions with two or three sections, and apply two sets of sections on each slide (one will be used for the experiment and the other will serve as a control). The water area should be larger than the size of the ribbon, so the sections have room to spread and even out. Optionally you can drain excess water from the slides using filter paper. Leave the sections on the hot plate overnight to dry and attach to the slides. Store them in slide boxes on room temperature or in the refrigerator at 4 °C.

  6. Feulgen staining
    1. The Feulgen reaction (Feulgen and Rossenbeck, 1924) quantitatively stains DNA. The nuclei become purple, while the rest of the cell is clear. The staining is done in Coplin jars (the volume of staining solution is 40 ml). Sections on objective slides are dewaxed in xylene, rehydrated through an ethanol series to water, hydrolyzed in 5 M HCl for 75 min at 20 °C, stained with Feulgen reagent for 120 min at 20 °C, washed for 45 min in six changes of SO2-water, dehydrated in an ethanol series, then mounted in DPX mounting medium.
    2. Feulgen staining protocol (adapted from Greilhuber and Ebert, 1994)

    Rehydration
    100% xylene
    RT
    Date of solution preparation:

    100% xylene
    5 min/step
    Feulgen:

    absolute ethanol

    HCl:

    96% ethanol

    SO2 water:

    70% ethanol

    Xylene:

    30% ethanol

    Ethanols:

    distilled water






    Hydrolysis
    5 M HCl
    20 °C
    75 min for FAA fixed

    (in a water bath)
    ____ min
    Paraplast embedded



    samples

    distilled water
    4 °C


    (cold water stops hydrolysis)
    5 min





    Feulgen
    Feulgen
    20 °C

    Staining
    (water bath)
    120 min









    Washing
    SO2 water
    RT
    250 ml SO2 water

    (work in fume hood!)
    3 x 2 min
    247.5 ml dH2O


    2 x 10 min
    2.5 ml 5 M HCl


    20 min
    1.25 g K2S2O2




    Dehydration
    distilled water
    RT


    30% ethanol
    5 min/step


    70% ethanol



    96% ethanol



    absolute ethanol



    100% xylene



    100% xylene






    Mounting
    DPX + cover slip


Recipes

  1. FAA (200 ml)
    100 ml 95% ethanol
    70 ml dH2O
    20 ml 37% formaldehyde solution
    10 ml glacial acetic acid
    Store at 4 °C
  2. Dehydrating solutions (for 100 ml each)
    10 ml TBA, 40 ml 95% ethanol, 50 ml dH2O
    20 ml TBA, 50 ml 95% ethanol, 30 ml dH2O
    35 ml TBA, 50 ml 95% ethanol, 15 ml dH2O
    55 ml TBA, 45 ml 95% ethanol
    75 ml TBA, 25 ml 95% ethanol
    100 ml TBA
    100 ml TBA
    100 ml TBA
  3. Schiff reagent (Feulgen stain)
    Boil 800 ml of dH2O. Add 4 g of pararosaniline hydrochloride and dissolve while mixing. Cool to 50 °C. Filter the solution through 2 glass fiber filters using vacuum. Add 120 ml 1 M HCl and 12 g K2S2O5. Leave the solution overnight in the dark at room temperature. Add 2 to 4 g decolorizing charcoal in the solution and mix. Filter the solution through 2 glass fiber filters using vacuum into a dry bottle (the stain must be clear and colorless). Store Schiff reagent in the refrigerator at 4 °C for up to 1 year (use until precipitate starts forming in the reagent)

Acknowledgments

The author acknowledges the Slovenian Research Agency for funding support.

References

  1. Bernardi, J., Lanubile, A., Li, Q. B., Kumar, D., Kladnik, A., Cook, S. D., Ross, J. J., Marocco, A. and Chourey, P. S. (2012). Impaired auxin biosynthesis in the defective endosperm18 mutant is due to mutational loss of expression in the ZmYuc1 gene encoding endosperm-specific YUCCA1 protein in maize. Plant Physiol 160(3): 1318-1328.
  2. Dermastia, M., Kladnik, A., Dolenc Koce, J. and Chourey, P. S. (2009). A cellular study of teosinte Zea mays subsp. parviglumis (Poaceae) caryopsis development showing several processes conserved in maize. Am J Bot 96(10): 1798-1807.
  3. Feulgren, R. and Rossenbeck, H. (1924). Mikroskopisch-chemischer Nachweis einer Nucleinsaure vom Typus der Thymonucleinsaure und die-darauf beruhende elektive Farbung von Zellkernen in mikroskopischen Praparaten. Hoppe-Seyler′s Zeitschrift für physiologische Chemie 135(5-6): 203-248.
  4. Greilhuber, J. and Ebert, I. (1994). Genome size variation in Pisum sativum. Genome 37(4): 646-655.
  5. Kladnik, A., Chourey, P. S., Pring, D. R. and Dermastia, M. (2006). Development of the endosperm of Sorghum bicolor during the endoreduplication-associated growth phase. J Cereal Sci 43(2): 209-215.
  6. Vilhar, B., Kladnik, A., Blejec, A., Chourey, P. S. and Dermastia, M. (2002). Cytometrical evidence that the loss of seed weight in the miniature1 seed mutant of maize is associated with reduced mitotic activity in the developing endosperm. Plant Physiol 129(1): 23-30.

材料和试剂

  1. 玉米籽粒
    在不同的发育阶段; 籽粒在授粉后最多20天(DAP)通常固定和切片良好,在后期阶段,胚乳逐渐干燥和硬,导致切片在切片机上切片时碎裂和撕裂
  2. Paraplast Plus(Pelco,目录号:18393; Sherwood Medical,目录号:8889-502005;或Sigma-Aldrich,目录号:P3683)
  3. 冰乙酸(Sigma-Aldrich,目录号:A6283)
  4. 甲醛溶液(组织学级,在H 2 O中37重量%)(Sigma-Aldrich,目录号:533998)
  5. 乙醇(96%和绝对)
  6. 叔丁醇(TBA)(Sigma-Aldrich,目录号:B85927)
  7. 对硝基苯胺盐酸盐(Sigma-Aldrich,目录号:P3750)
  8. 偏亚硫酸氢钾(K 2 SS 2 O 5)(Sigma-Aldrich,目录号:31268)
  9. 二甲苯(二甲苯,组织级)(Sigma-Aldrich,目录号:534056)
  10. 5M HCl(Merck-Millipore,目录号:1099110001)
  11. 脱色炭(BDH,GB)(Sigma-Aldrich,目录号:161551)
  12. DPX封固剂(Sigma-Aldrich,目录号:06522)
  13. 甲醛 - 乙酸 - 乙醇(FAA)(参见配方)
  14. 脱水解决方案(参见配方)
  15. 席夫试剂(见配方)

设备

  1. 玻璃小瓶(体积〜20ml)
  2. 玻璃纤维过滤器(Microfibre过滤器,Whatman GF/C)
  3. 手术刀,剃刀刀片
  4. 金属镊子(使用专用镊子蜡工作,他们很难清洗)
  5. 一次性塑料巴斯德移液器
  6. 真空干燥器(Bel-Art,型号:420100000)
  7. 染色皿(用于熔融Paraplast Plus)(Electron Microscopy Sciences,目录号:70312-21)
  8. 热板(设置在低 - 约68°C),与铝箔帐篷,以保持热在内部
  9. 酒精燃烧器
  10. 塑料或金属模具
    一次性基础模具(Pelco,目录号:27147)
    Tissue-Tek Base Mold(Sakura,目录号:4123)
  11. 组织包埋盒基部(Pelco,目录号:27168-1)或嵌入环(Pelco,目录号:27169-1)或Tissue-Tek嵌入环(Sakura,目录号:4151)
  12. 带有熔融Paraplast Plus(专用于蜡工作)的小烧杯
  13. 托盘上有一薄层冷水(通过加冰冷却)
  14. 冰桶里装满冰
  15. 平冰袋(冷冻冷藏包)或黄铜板(可选)
  16. 旋转切片机
  17. 一次性切片机刀片
  18. 小刷子
  19. 尖端钳子
  20. 单刃剃刀刀具
  21. 深色纸板板
  22. 立体显微镜
  23. Coplin罐(Electron Microscopy Sciences,目录号:70316-02)

程序

  1. 固定在FAA
    1. 将玻璃小瓶中的FAA固定剂(〜20ml小瓶,10ml固定剂)。 保持小瓶在冰上,固定剂应该是冷的,当您放置组织。用手术刀或剃刀刀片切成片,至少有一个维度小于2或3毫米,立即浸入冷固定剂。
      注意:切割组织时,请考虑您希望获得的切片类型。
    2. 在真空干燥器中放置具有固定材料的开放小瓶,并将其暴露于中等真空15分钟以将空气从组织中抽出。 如果组织不下沉,当你释放真空,应用和释放真空一次。 抽真空后,用新鲜固定剂替换小瓶中的固定剂
    3. 在4°C(冰箱)固定过夜。

  2. 脱水在一系列TBA中
    1. 在使用前将纯TBA(1 L)放在温暖的地方(例如在孵化炉顶部),它在低于25°C时冻结。准备脱水解决方案和存储解决方案a - e室温(RT)和f - h在孵化炉。
    2. 根据组织尺寸(6DAP玉米粒半天,每个步骤中12天DAP玉米粒,每个步骤1天)将每个步骤中的水分组织脱水1小时至一天。脱水溶液a - e步骤应在RT,步骤f - h在孵化炉中在56-60°C。
      使用塑料巴斯德吸管从样品瓶中取出旧的溶液,然后更换等量的下一个溶液(与您的样品相比,您需要相对更大量的脱水溶液)。在小瓶上的标签上标记脱水进度。或者,您可以将旧溶液从样品瓶中倒出,更换新样品(您不需要完全排出样品瓶)。在通风橱工作。在TBA的最后一次更换中,将样品瓶仅填充到样品瓶体积的一半,以便为下一步中添加Paraplast Plus留出足够的空间。

  3. 石蜡包埋(Paraplast Plus)
    1. 保持熔融Paraplast Plus和塑料移液管在孵化炉中在56-60°C。您需要大量的熔融石蜡,因为熔化需要很长时间(例如两个长方形的组织学染色盘,容纳〜300ml)。
    2. 在半充满TBA的小瓶中加入Paraplast Plus蜡的小丸。或者,您可以向小瓶中的TBA添加等体积的熔融蜡。 Paraplast Plus具有精细的结构和添加剂(DMSO),有助于更好地渗入组织,这对于无故障切片非常重要。保持在56-60°C烤箱,与瓶盖连接和混合几次,当蜡完全融化。在烘箱中过夜。
    3. 第二天,将TBA /蜡混合物倒入或吸取到通风橱中的废物容器中(您可以用铝箔制作一个小的废物容器)。加入新鲜的熔融蜡,并放置在烤箱中过夜,小瓶打开,使所有的TBA蒸发。第二天用新鲜的蜡更换几次(使用温热的移液管或倒出),例如每天两次,两天。
  4. 铸蜡块
    1. 在实验室中设置较暖的空气条件(如果太冷,蜡会固化太快)
    2. 设备:
      1. 热板(设置在低 - 约68°C),与铝箔帐篷,以保持热在内部
      2. 金属镊子(使用专用镊子蜡工作,他们很难清洗)
      3. 酒精燃烧器
      4. 塑料或金属模具
      5. 嵌入戒指或嵌入底座
      6. 一个带有熔融Paraplast Plus(也必须专门用于蜡工作)的小烧杯
      7. 一个带有薄层冷水的托盘(通过加冰冷却)
      8. 一个装满冰的冰桶
      9. 平冰袋(冷冻冷藏包)或黄铜板(可选)
    3. 保持少量的熔融蜡在烧杯中的铝板帐篷在热板。 我们还保护热板与一层铝箔,以保持它清洁蜡。 如果板没有精确地热调节,则始终检查板温度 - 如果板太热而不能接触,则必须关闭板,然后当蜡开始凝固时将其打开。 长期温度超过62°C会损坏Paraplast Plus。 此外,如果使用金属模具,请在使用之前将它们放在热板上
    4. 取一个小瓶,样品一次从烤箱中取出,并将其放在热板上。使用铅笔或防水标记标记与样本数量相同数量的嵌入环。在模具中倒一些蜡,取出一个样品从小瓶中用在酒精燃烧器的火焰温暖的镊子。
      注意:小心,只是通过镊子通过火焰很短时间,如果烟雾从镊子,他们太热了。
      考虑您希望以后获得的节的类型,在加热板上的模具中定位样本。小心地将模具转移到黄铜板/冰包上或只是在桌子上,以使蜡的底层固化并将样品固定就位。如果样品难以在热板上定位,那么当蜡底层是固体时,您现在可以定位样品。填充模具的熔融蜡到边缘,并盖上标记的嵌入环。然后用冷水和冰将模具转移到托盘中,并在那里保持约10分钟,使蜡完全固化。然后将模具放在冰上,等待几分钟,然后从模具中取出蜡块(用冰冷却有助于块从模具中松脱)。将模具放回热板上重新使用。我们通常一次处理5到10个块。将块放在冰箱中4°C(见图1)。


      图1.在石蜡中嵌入组织样本的工作场所组织示意图
  5. 切片
    1. 设备:
      1. 旋转切片机
      2. 一次性切片机刀片
      3. 一个小刷子
      4. 具有尖端的镊子
      5. 单刃剃刀刀片
      6. 深色纸板(用于放置和观察部分)
      7. 立体显微镜(可选,但推荐)
      8. 热板设置在40°C
      9. 用蒸馏水
        小瓶
      10. 塑料移液器
      11. 清洁的物镜显微镜载玻片,磨砂在一个边缘
      12. 铅笔
    2. 使用单刃刀片修整样品周围的蜡块,使得上边缘和下边缘平行,并且左边缘和右边缘成一定角度,使得块的下边缘比上边缘长。此外,块的侧面不应该是垂直的,而是倾斜的 - 总是用剃刀刀片从样品的顶部到底部以干净的切割切割块(参见图2)。


      图2.切割石蜡包埋的组织样品。 顶部:修剪样品周围的石蜡的方向。底部:在切片机上切片并将切片布置在载玻片上。

    3. 将样品的嵌入环固定在切片机上的样品架中。设置切片厚度(通常为10至20μm)和切割角度(约7度)。开始剖分;前几个部分不会好,所以用小刷子去除它们,总是远离刀片​​,不要朝向它!当样品的表面变平时,色带将开始形成。切割足够的部分,以适合他们在一个黑色的纸板,用刀片切割丝带。剪切几条丝带,直到你确定你已经切割感兴趣的区域,然后用立体显微镜观察部分,以找到有趣的部分(内核的中间纵截面)。< br />
    4. 用铅笔标记物镜载玻片,将它们放在热板(40℃)上,并在其上涂一些水(几乎覆盖它们)。在两个或三个部分的较小区域中剪切色带,并在每个幻灯片上应用两组部分(一个用于实验,另一个用作控制)。水面积应大于色带的尺寸,因此部分有扩展的空间,甚至可以伸出。 (可选) 可以使用滤纸从幻灯片中排出多余的水。离开热板上的部分过夜干燥,并附加到幻灯片。将它们储存在室温的滑动盒中或4℃的冰箱中
  6. Feulgen染色
    1. Feulgen反应(Feulgen和Rossenbeck,1924)定量染色DNA。核变成紫色,而细胞的其余部分是清楚的。染色在Coplin瓶中进行(染色溶液的体积为40ml)。将目标载玻片上的切片在二甲苯中脱蜡,通过乙醇系列再水合成水,在20℃下在5M HCl中水解75分钟,在20℃下用Feulgen试剂染色120分钟,在六次更换SO 2 - 水,在乙醇系列中脱水,然后安装在DPX封固剂中。
    2. Feulgen染色方案(改编自Greilhuber和Ebert,1994)
    再水合
    100%二甲苯
    RT
    溶液准备日期:

    100%二甲苯
    5分钟/步
    Feulgen:

    无水乙醇

    HCl:

    96%乙醇

    SO 2 水:

    70%乙醇

    二甲苯:

    30%乙醇

    乙醇:

    蒸馏水






    水解
    5 M HCl
    20°C
    75分钟为FAA固定

    (在水浴中)
    ____ min
    Paraplast嵌入式



    样品

    蒸馏水
    4°C


    (冷水停止水解)
    5分钟





    Feulgen
    Feulgen
    20°C

    染色
    (水浴)
    120分钟









    洗涤
    SO 水
    RT
    250ml SO 2水

    (在通风橱工作!)
    3 x 2分钟
    247.5ml dH 2 O


    2 x 10分钟
    2.5ml 5M HCl


    20分钟
    1.25g K sub 2 S sub 2 O sub 2/




    脱水
    蒸馏水
    RT


    30%乙醇
    5分钟/步


    70%乙醇



    96%乙醇



    无水乙醇



    100%二甲苯



    100%二甲苯






    安装
    DPX +盖玻片


食谱

  1. FAA(200ml)
    100ml 95%乙醇 70ml dH 2 O 2 / 20ml 37%甲醛溶液
    10ml冰乙酸 存储在4°C
  2. 脱水溶液(每次100ml)
    10ml TBA,40ml 95%乙醇,50ml dH 2 O 2 / 20ml TBA,50ml 95%乙醇,30ml dH 2 sub 35ml TBA,50ml 95%乙醇,15ml dH 2 sub 55ml TBA,45ml 95%乙醇 75ml TBA,25ml 95%乙醇 100 ml TBA
    100 ml TBA
    100 ml TBA
  3. 希夫试剂(Feulgen stain)
    煮沸800ml dH 2 O。 加入4g盐酸副玫瑰苯胺并在混合下溶解。 冷却至50℃。 使用真空通过2个玻璃纤维过滤器过滤溶液。 加入120ml 1M HCl和12g K 2 S 2 O 5 O 5。 将溶液在室温黑暗中保持过夜。 在溶液中加入2〜4g脱色炭并混合。 使用真空将溶液通过2个玻璃纤维过滤器过滤到干燥的瓶子中(染色必须是透明和无色的)。 将希夫试剂在4℃的冰箱中保存长达1年(使用直至试剂中开始形成沉淀)

致谢

作者承认斯洛文尼亚研究机构的资金支持。

参考文献

  1. Bernardi,J.,Lanubile,A.,Li,Q.B.,Kumar,D.,Kladnik,A.,Cook,S.D.,Ross,J.J.,Marocco,A.and Chourey,P.S。(2012)。 缺陷型胚乳18突变体中受损的生长素生物合成是由于ZmYuc1中突变的表达缺失基因编码玉米中胚乳特异性YUCCA1蛋白。 植物生理学 160(3):1318-1328。
  2. Dermastia,M.,Kladnik,A.,Dolenc Koce,J.and Chourey,P.S.(2009)。 teosinte的细胞研究Zea mays subsp。 (禾本科)颖果发育显示在玉米中保守的几个过程。 96(10):1798-1807。
  3. Feulgren,R。和Rossenbeck,H。(1924)。 Mikroskopisch-chemischer Nachweis einer Nucleinsaure vom Typus der Thymonucleinsaure und die-darauf beruhende elektive Farbung von Zellkernen in mikroskopischen Praparaten。 Hoppe-Seyler's Zeitschriftfürphysiologische Chemie 135 6):203-248
  4. Greilhuber,J。和Ebert,I。(1994)。 豌豆豌豆中的基因组大小变化。 基因组 37(4):646-655
  5. Kladnik,A.,Chourey,P.S.,Pring,D.R。和Dermastia,M。(2006)。 在endoreduplication相关生长阶段期间开发高粱双色胚乳 。 J Cereal Sci 43(2):209-215。
  6. Vilhar,B.,Kladnik,A.,Blejec,A.,Chourey,P.S.and Dermastia,M。(2002)。 细胞计数证据表明玉米miniature1种子突变体中种子重量的损失与有丝分裂活性降低有关 在发育的胚乳中。植物生理学 129(1):23-30。
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。

X


How to cite this protocol: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Kladnik, A. (2013). Maize Kernels – Fixation in FAA, Embedding, Sectioning and Feulgen Staining. Bio-protocol 3(15): e835. DOI: 10.21769/BioProtoc.835; Full Text
  2. Bernardi, J., Lanubile, A., Li, Q. B.,Kumar, D., Kladnik, A., Cook, S. D., Ross, J. J., Marocco, A. and Chourey, P.S. (2012). Impaired auxin biosynthesis in the defective endosperm18mutant is due to mutational loss of expression in the ZmYuc1 geneencoding endosperm-specific YUCCA1 protein in maize. Plant Physiol 160(3): 1318-1328.




  3. 可重复性反馈:

    • 添加图片
    • 添加视频

    我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

    问题&解答:

    • 添加图片
    • 添加视频

    (提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。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或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


    登陆 | 注册
引用格式
分享
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook