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Nov 2017

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Visualization of Plant Cell Wall Epitopes Using Immunogold Labeling for Electron Microscopy
利用电子显微镜免疫金标记法进行植物细胞壁表位的可视化观察   

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Abstract

Plant cell walls consist of different polysaccharides and structural proteins, which form a rigid layer located outside of the plasma membrane. The wall is also a very dynamic cell composite, which is characterized by complex polysaccharide interactions and various modifications during cell development. The visualization of cell wall components in situ is very challenging due to the small size of cell wall composites (nanometer scale), large diversity of the wall polysaccharides and their complex interactions. This protocol describes immunogold labeling of different cell wall epitopes for high-resolution transmission electron microscopy (TEM). It provides a detailed procedure for collection and preparation of plant material, ultra-thin sectioning, specimen labeling and contrasting. An immunolabeling procedure workflow was optimized to obtain high efficiency of carbohydrates labeling for high-resolution TEM. This method was applied to study plant cell wall characteristics in various plant tissues but could also be applied for other cell components in plant and animal tissues.

Keywords: Cell walls (细胞壁), Cell wall epitopes (细胞壁表位), Polysaccharides (多糖), Immunogold labeling (免疫金标记), Transmission electron microscopy (透射电子显微镜)

Background

Plant biomass is mainly composed of cell walls, which are widely used as an energy source in our daily life (Loqué et al., 2015). At the microscopic scale, cell walls consist of cellulose microfibrils embedded in complex matrix polysaccharides (hemicelluloses, pectins) and structural proteins. Cellulose microfibrils (CMFs) are the largest wall polymers with a radius of 3-5 nm and many micrometers long (Cosgrove, 2005). The orientation of CMFs determines the direction of growth and cell anisotropy (Baskin, 2005), but the CMFs also interact with other wall components all together modifying the wall properties (reviewed in Majda, 2018; Majda and Robert, 2018). The study of cell wall composition has a long history, going back to when different chemicals were applied to bind to the wall composites; however, many of them had a wide range of targets (Wallace and Anderson, 2012; Voiniciuc et al., 2018) and could be observed only via light microscope resolution. In contrast, the immunogold labeling is characterized by a high specificity of antibodies and high-resolution imaging, which can precisely localize the wall epitopes across the wall matrix (e.g., Majda et al., 2017). Despite electron microscopy being a relatively old method, it is not broadly used for plant cell walls. The reason could be that it is time-consuming, requires long training and extensive preparation time. In this protocol, I will walk you through all the steps concerning sample preparation, specificity for all the reagents and troubleshooting.

Materials and Reagents

  1. Adhesion slides, Polysine, 25 x 75 x 1.0 mm (VWR, catalog number: 631-0107)
  2. Aluminum foil
  3. Centrifuge tubes 15 ml (PluriSelect, catalog number: 05-00002-01)
  4. Centrifuge tubes 50 ml (sterile) (PluriSelect, catalog number: 05-00001-01)
  5. Compressed air in the can (e.g., air duster PRF 4-44)
  6. Disposable pH indicator paper (universal indicator paper) (Johnson, catalog number: 101.3C)
  7. Disposable plastic Pasteur pipettes (BRAND, catalog number: 747750)
  8. Double-edged razor blades (Personna, catalog number: 171930)
  9. Embedding capsules (8 mm flat, polypropylene capsules) (TAAB, catalog number: C095)
  10. Filter papers (circles, 150 mm Ø), (Whatman, catalog number: 1001150)
  11. Glass vials with plastic snap-cap (ca. 41 x 24 mm) (Karl Hecht, catalog number: 2783/3), or clear glass vials with snap-cap (closed top, PE transparent, 18 mm, 500 ml 20 x 40 mm) (VWR, catalog number: 548-0555)
  12. Grids for transmission electron microscopy, e.g., grid size 100 mesh x 250 μm pitch, nickel or copper (TAAB, maxtaform HF4, catalog number: GM021N; Sigma-Aldrich, catalog number: G1528; Agar Scientific, catalog number: G2500C)
  13. Light-duty 3-Ply tissue wipers (VWR, catalog number: 82003824-CS)
  14. Metal needle or dissection needle (VWR, catalog number: 10806-330)
  15. Microcentrifuge tubes 1.5 ml (Sigma-Aldrich, catalog number: Z606340)
  16. Microscope slides, 25 x 75 x 1.0 mm (VWR, catalog number: 48300-025 or Thermo Scientific, catalog number: 10144633CF)
  17. Paper tags
  18. Parafilm (Sigma-Aldrich, catalog number: P7543)
  19. Pencil
  20. Pipette tips
  21. Plastic Petri dishes (Fisherbrand, catalog number: S33580A)
  22. Protective gloves (Honeywell, catalog number: Dermatril 740)
  23. Round silicone rubber (TAAB, catalog number: G082)
  24. Single edge razor blades with aluminum spine (VWR, catalog number: 233-0156)
  25. Slide labels (Agar Scientific) or one side adhesive paper
  26. Square Petri dish (120 mm) (Corning, catalog number: BP124-05)
  27. Transparent tape (e.g., scotch)
  28. Waste containers
  29. Agar, plant agar (Duchefa Biochemie, catalog number: 9002-18-0)
  30. Bovine Serum Albumin (BSA), lyophilized powder ≥ 96% (agarose gel electrophoresis) (Sigma-Aldrich, catalog number: 9048-46-8)
  31. Disodium phosphate (Na2HPO4) (store in ambient temperature: room temperature, approx. 21 °C) (Sigma-Aldrich, catalog number: 7558-79-4)
  32. Distilled water
  33. Ethanol laboratory reagent, absolute, ≥ 99.5% (flammable, stored in designated place) (Sigma-Aldrich, catalog number: 64-17-5)
  34. Formaldehyde (FA) (10 ml ampule of 16% methanol-free FA) (health hazards, store closed in ambient temperature, or open in cold room/refrigerator 4 °C) (Thermo Scientific, catalog number: 28908) or paraformaldehyde (PFA) for histology (CH2O)n, (store in cold room/refrigerator 4 °C) (J.T. Baker, catalog number: S898-07)
  35. Formvar solution (1% formvar in dichloroethane, for microscopy) (Sigma-Aldrich, catalog number: 63148-64-1)
  36. Glutaraldehyde (GA) (grade I, 1 ml ampule of 25% GA in H2O, specially purified for use as an electron microscopy fixative, linear formula: OHC(CH2)3CHO (health hazards, store closed in ambient temperature, or open in cold room/refrigerator 4 °C) (Sigma-Aldrich, catalog number: 111-30-8)
  37. Hydrogen chloride (HCl) (Sigma-Aldrich, catalog number: 7647-01-0)
  38. LR white resin medium grade–catalyzed (health hazards, store in cold room/refrigerator 4 °C) (TAAB, catalog number: L012)
  39. Monopotassium phosphate (KH2PO4) (Sigma-Aldrich, catalog number: 7778-77-0)
  40. Monosodium phosphate (NaH2PO4) (store in ambient temperature) (Sigma-Aldrich, catalog number: 7558-80-7)
  41. Murashige and Skoog basal medium (MS) (Sigma-Aldrich, catalog number: M5519)
  42. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: 7447-40-7)
  43. Primary antibodies (PlantProbes: www.plantprobes.net or the University of Georgia: www.ccrc.uga.edu)
  44. Secondary antibodies, e.g., EM Goat anti-Rat IgG (H+L): 10 nm Gold (BBI Solutions, catalog number: 014990) or EM Goat anti-Mouse IgG (H+L) 10 nm Gold (BBI Solutions, catalog number: EM.GMHL10)
  45. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 7647-14-5)
  46. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 1310-73-2)
  47. Sucrose (Sigma-Aldrich, catalog number: 57-50-1)
  48. Toluidine blue for microscopy (Sigma-Aldrich, catalog number: 6586-04-5)
  49. TopVision low melting point agarose (LMP agarose) (store in ambient temperature) (Thermo Scientific, catalog number: R0801)
  50. Tween 20 (Signa-Aldrich, catalog number: P1379)
  51. Uranyl acetate (UA) (health hazardous, store in ambient temperature) (VWR, catalog number: 541-09-3)
  52. Half strength MS basal medium (see Recipes)
  53. Paraformaldehyde-glutaraldehyde (4% PFA and 0.05% GA) fixative solution (see Recipes)
  54. Phosphate buffer (PB), 0.1 M solution (pH = 7.2) (see Recipes)
  55. Low melting point (LMP) agarose 1% solution (see Recipes)
  56. Different ethanol concentrations 10%-95% (see Recipes)
  57. Different LRW resin concentrations 10%-75% (see Recipes)
  58. Toluidine blue solution (see Recipes)
  59. Blocking Reagent (BR) 1% (see Recipes)
  60. Antibodies solutions (see Recipes)
  61. Phosphate Buffered Saline (PBS), 0.1 M solution (pH = 7.2) (see Recipes)
  62. Uranyl acetate solution (5%) (see Recipes)

Equipment

  1. Analytical balance
  2. Autoclave (YPO, model: D66161)
  3. Centrifuge (Marshall Scientific, Eppendorf, model: 5417C)
  4. Conical flask
  5. Desiccator (Thermo Fisher, model: 5311-0250)
  6. Diagonal cutting pliers e.g., stanley diagonal cutting pliers, 5 (Robosource, catalog number: 15-11)
  7. Diamond knife, e.g., ultra 45° (Diatome)
  8. Flat beaker or crystallizer (with a wide diameter)
  9. Forceps
  10. Freezer (-20 °C)
  11. Fume hood
  12. Glass baker (50 ml)
  13. Glass knife strips (Agar Scientific, catalog number: AGG336)
  14. Glass knifemakers for histology knives ( LKB, catalog number: LKB 7801B; or Agar Scientific, catalog number: AGL4158)
  15. Grid storage box (LKB/Leica catalog number: G133, or TAAB Gilder G062)
  16. Lab oven/incubator (60 °C)
  17. Light microscope
  18. Magnetic hotplate stirrer with magnetic stir bar
  19. Metal 1.5 ml Eppendorf rack
  20. Microtome (Reichart Ultracut)
  21. Microwave oven
  22. pH meter
  23. Pipettes
  24. Protective clothes and mask
  25. Refrigerator (4 °C)
  26. Rotary shaker or orbital shaker (IKA KS 130 Basic)
  27. Slide drying rack or slide staining jar (e.g., DWK Life Sciences Wheaton)
  28. Small bench clamp workshop
  29. Thin painting brush
  30. Transmission Electron Microscope (JEOL, model: JEM-1230)
  31. Tweezers: negative-action style: thin curved tips (Dumont, catalog number: 0203-N7-PO) or thin tips (Dumont, catalog number: 0302-N0-PO-1)
  32. Tweezers: straight with Geneva pattern, thin tips (Dumont, catalog number: 0103-0-PO)
  33. Vortex mixer (Vortex-Genie 2)
  34. Warming plate, or slide drying hotplate (Agar Scientific, model: AGL4384) or spirit lamp burner

Procedure

  1. Plant material fixation
    In this section, I describe the procedure for plant material preparation and fixation. This protocol was developed for Arabidopsis thaliana leaves, but it could also be applied to other organisms and tissues such as roots, shoots and woody tissues in tree species.
    1. Sterilize the seeds before sowing
      1. Place a small number of seeds into 1.5 ml Eppendorf tubes (approx. 5% of the Eppendorf tube volume).
      2. Add 1 ml of 70% ethanol with Tween 20 for 2 min.
      3. Replace 70% ethanol and Tween 20 with 1 ml of 95% ethanol for 1 min.
      4. Remove the ethanol and wait for the seeds to dry.
      Note: Perform the seeds sterilization under a sterile fume hood.
    2. Grow Arabidopsis seedlings for 2 weeks on vertical agar plates (Recipe 1) in the chamber with long day condition (16 h) (temperature 20 °C and 18 °C at day and night, respectively).
      Note: To synchronize the growth, vernalize the seeds by keeping the plates in a cold room/refrigerator at 4 °C for 2-3 days (in darkness).
    3. Harvest plant material and place it directly in the vials filled with 3-5 μl of cold paraformaldehyde-glutaraldehyde (PFA-GA) fixation solution (Recipe 2).
      Note: Remember to harvest the same leaf number from each plant (counting from the bottom to the top: cotyledons, leaf 1, leaf 2, leaf 3, leaf 4, meristem). To allow the fixative to penetrate well, cut small squares (~2 mm2 max). in the middle part of the leaf. Harvest at least 10 leaves from different plants. Conduct all the fixation steps under the fume hood with protective gloves and clothes (for handling restrictions see Safety Data Sheet SDS provided by the retailer). Mark the vials by writing the names with a permanent marker and sticking a transparent tape on these labels.
    4. Vacuum samples in a desiccator at ambient temperature until plant pieces will sink (approx. 4 h).
      Note: It might happen that some of the samples are floating after vacuuming, which indicates that samples more likely contain oxygen. Try to collect samples, which are at the bottom of the vials.
    5. Place the vials on a rotary or orbital shaker and leave the samples mixing for a couple of hours.
    6. Keep the samples in a cold room/refrigerator (4 °C) overnight.
    7. Discard PFA-GA fixative.
    8. Wash the samples with phosphate buffer (PB) (Recipe 3) (twice for 30 min each).
      Note: Discard PFA-GA fixative and first PB washing by using disposable plastic Pasteur pipettes in the assigned waste bottle. Wash by adding at least 5 ml of PB or distilled water (the more PB and distilled water, the better the washing). Conduct all washing steps slowly mixing on rotary or orbital shaker under a fume hood.

  2. Embedding plant material in low melting point (LMP) agarose
    Embedding small plant pieces in LMP agarose is the best way to orient the sample for sectioning (e.g., cross or longitudinal). It marks the direction of shoot/root tip (Figure 1A) or the localization of abaxial and adaxial leaf sides (Figure 1B). Embedding also facilitates the handling and protection of samples.


    Figure 1. Orienting the plant pieces in low melting point (LMP) agarose. A-B. Samples embedded in cuboid-shaped agarose blocks presenting one cut edge to mark the orientation of the sample (e.g., top left in Figure 1A). A. Top part of the root is marked by a cut edge. B. The adaxial side of the leaf is in the front when the cut edge is on the top left.

    1. Wash the samples with distilled water (twice for 30 min each).
    2. Pour a thin (3-5 mm thick) layer of agarose (Recipe 4) on a square plastic Petri dish and wait a few minutes until agarose cools down (but should not solidify).
    3. Place the plant material on agarose by using tweezers (many pieces on the same plate but keep distance between the samples approx. 2-3 cm2).
      Note: Handle the plant specimens with care not to damage the tissues.
    4. Add more agarose (± 3 mm thick layer) to cover the plant specimens (wait until agarose solidifies) (Figure 2A).
    5. Cut off small cubes of agarose with the embedded specimens (one specimen in one cube) (Figure 2B).
      Note: The size of agarose cubes should not be bigger than the diameter of embedding capsules (8 mm Ø).
    6. Cut one of the corners in the agarose cube to orient the sample (Figures 1, 2B and 2C).
    7. Move the blocks to vials filled with distilled water, then discard the water before proceeding to the next step.


      Figure 2. Embedding the plant material in agarose. A. Square Petri dish filled with a layer of 3 mm thick agarose. B. Small cuboid-shaped agarose blocks with cut one of the edges (all the leaf pieces are seen from the adaxial side). C. Magnified agar cube with the abaxial side of the leaf in the front. In this case, big plant pieces were used for visualization, but they should be ± 5 times smaller.

  3. Dehydrating specimens embedded in agarose blocks
    Here I describe a common method to remove water from samples and to enable better penetration of resin. Time intervals can be adjusted; however, longer intervals give better quality embedding. Ideally, the dehydration process would be carried over the span of two days, but all dehydration steps can also be carried out within the same day (depends on the sample size).
    1. Dehydrate the samples in a graded ethanol series as below (Recipe 5):
      10% ethanol (twice for 30 min each)
      20% ethanol (twice for 30 min each)
      30% ethanol (twice for 30 min each)
      50% ethanol (twice for 30 min each)
      Note: The amount of ethanol added varies according to the size and number of the samples embedded in agarose. Make sure that agarose blocks are completely covered by ethanol.
    2. Leave the samples in 50% ethanol in ambient temperature (on the bench) overnight.
      Note: Close the vials with plastic snap-cap to prevent ethanol evaporation.
    3. Continue to dehydrate the samples in a graded ethanol series as below:
      70% ethanol (twice for 30 min each)
      80% ethanol (twice for 30 min each)
      90% ethanol (twice for 30 min each)
      95% ethanol (three times for 30 min each)
      99.5% ethanol (three times for 30 min each)
      Note: Carry all dehydration steps on rotary or orbiter shaker. The vials should be closed with plastic snap-caps. Some intermediate ethanol dilutions can be omitted (e.g., 20% and 80% ethanol), but it might affect the quality of embedding. The volume of alcohol in the last step should be the same for all vials.

  4. Resin embedding
    In this step, alcohol is replaced with viscous and low soluble LR white (LRW) resin. All these steps must be carried gradually with vigorous mixing of the samples on an orbital shaker. Perform all these steps in cold room/refrigerator (4 °C) to prevent resin polymerization.


    Figure 3. Embedding the plant material in resin. A. Embedding capsule and paper tags. B. Capsule after resin polymerization. C. Capsule cut along to remove the resin block. D. Resin block alone. E. Trimmed resin block into a trapezoid shape.

    1. Slowly add a few drops of resin to the vials with a known volume of 99.5% ethanol until the resin content will reach 10% (v/v) of the overall volume.
      Note: All steps, including the exchange of the resin, must be carried out using protective gloves and clothes under the fume hood. Keep the vials closed with the plastic snap-caps mixing in the orbital shaker in cold room/refrigerator (4 °C). Always discard the resin to designated waste.
    2. Leave the vials in the orbital shaker to mix samples overnight.
    3. Exchange resin 10% in alcohol with a new resin in a graded resin series as below (Recipe 6):
      10% resin (approx. 5 h)
      25% resin (approx. 5 h)
      50% resin overnight
      75% resin (approx. 5 h)
      100% resin overnight
      100% (fresh) resin overnight
      Note: Some intermediate resin dilutions can be omitted (e.g., 25% and 75%), but it might affect the quality of embedding.

  5. Closing samples in the embedding capsules and resin polymerization
    This is the last step of the embedding procedure, in which you must pay attention to properly orientate the samples (Figure 1). The agarose blocks should lie flat in the middle of the bottom part of the capsule (Figures 4A and 4B), which need to be oriented in a parallel direction towards the knife edge (see Procedure G, Figure 6). Handle the capsules with care to prevent the slipping of the samples close to the corners, which could cause some issues in trimming the sample and sectioning.


    Figure 4. Trimming the plant specimen prior sectioning. A-B. A capsule with a piece of the leaf embedded in resin (A). The sample should be in the middle of the bottom part of the capsule (B). C-E. Resin with specimen alone seen from the bottom: resin before trimming (C), the direction of trimming (D), trimmed specimen (E). F. Trimmed resin block seen from the side.

    1. Place the capsule (Figure 3A) on a rack and fill half of the capsule with resin.
    2. Put the sample in the capsule, orient the sample well and fill-up the capsule with fresh resin.
    3. Cut small paper tags, label them with a pencil and place them in the top of the capsules.
      Note: The capsule should be filled with resin so that, by closing it, no space for air bubbles is left. Use a pencil to tag your samples instead of a pen or a marker, which can be washed out by the resin.
    4. Place the capsules in the incubator/oven (60 °C) for 24 h (or until it becomes solid) to polymerize the resin.
      Note: Remember to check the temperature of the oven (60 °C). A temperature that is too low can affect the polymerization (resin will not polymerize equally), while a temperature that is too high or an excessively long incubation can lead to cracking or breaking of the resin.
    5. Samples are ready when the resin becomes stiff (Figure 3B).
      Note: To know if the resin solidified, indent a metal needle to the resin surface.

  6. Preparing the grids for transmission electron microscope (TEM)
    The samples for the TEM are mounted on a small grid, which is then inserted inside the TEM. The grid must be coated with formvar, which is an adhesive layer holding the sections. While preparing the grids, make sure that microscope slides, tweezers and laboratory glassware are clean (use tissue wipers and compressed air to clean the dust on the surfaces).
    1. Add approx. 50 ml of formvar solution to a small beaker (Figure 5A).
    2. Grab the microscope slide in a vertical position (holding a dull side) and vigorously dip approx. half of the slide in the formvar solution (no more than approx. 5 s in the solution) (Figure 5B).
      Note: The formvar will form a thin film on the side.
    3. Dry the slide with a film in a vertical position on a drying rack for 3-10 min.
      Note: Do not touch any part of the slide, which was in contact with the formvar solution.
    4. Prepare glassware with a wide diameter (≥ 10 cm) and fill it up with distilled water. Place the grids on a round silicone rubber or filter paper (Figure 5C).
    5. Scratch the slide edges with a sharp razor blade and blow moist air on the slide.
    6. Dip the slide vertically (90° or ≥ 45°) in the distilled water bath.
      Note: You will see the film detaching from the glass and then floating on the water surface.
    7. Using fine tweezers, put the grids onto a floating film with the dull side in contact with formvar (put as many grids as possible) (Figures 5D and 5E).
      Note: Each grid has two sides: a dull side and a shiny side (Figure 7A). Sections will be mounted on the formvar-coated dull side (this side should be in contact with buffers, antibodies, distilled water, contrasting stain) (Figure 7B), and the shiny side will be in contact with filter paper during the drying of the samples (it is up to you which side you want to coat with formvar and put the sections on but remember to be consistent).
    8. Prepare a new slide covered by one side adhesive paper or slide label (Figure 5F).
    9. Grab that slide in vertical position and touch one of the edges of the floating film (Figure 5G).
    10. Dip the slide into the water, which will cause adhesion of the film with grids to the slide (Figures 5H and 5I).
    11. Leave the slide with grids covered with formvar until dry.
      Note: Store the slides in a closed box or plastic Petri dish in cold room/refrigerator (4 °C).


      Figure 5. Preparing the grids for TEM. A. Baker filled with formvar solution and a slide on the slide staining jar. B. Dipping of the microscope slide in formvar solution. C. Glassware filled up with distilled water, Petri dish with round silicone rubber and grids on it, tweezers. D. Tweezers holding the outer ring of the grids. E. Grids on a floating formvar film with the dull side of the film. F. Sticking a slide label to the slide. G. Slide in vertically before touching the edges of the floating film. H. Dipping the slide in water causing the adhesion of film with grids to the slide. I. Slide with formvar and grids on it.

  7. Ultrathin sectioning for electron microscopy
    The most important thing in the preparation of the sections is to properly orient the sample. Remember to regularly control the sections quality. Make sure that the cells/tissue/organ is oriented in parallel to the knife (Figure 6) and if they are not, then correct the angle between the sample and the knife edge.


    Figure 6. Ultrathin sectioning for electron microscopy. Begin with orienting the specimen in parallel to the knife edge. When resin is close to the knife, a reflection of the knife will be seen on the surface of the resin. This reflection should be parallel to the sample (if properly oriented). With sectioning progression, make sure that the cells/tissue/organ are oriented in parallel to the knife (remember to correct the angle between the sample and the knife edge).

    1. Immobilize the capsules using a small bench clamp workshop.
    2. Remove the capsules using stiff single edge razor blades and diagonal cutting pliers (Figures 3C and 3D).
    3. Trim the bottom of the sample with a stiff single edge razor blade and then use thin double-edged razor blades for more precise cutting (Figures 3E and 4C-4F).
      Note: The bottom of the sample, which will be in parallel to the knife edge (see Procedure E), should be trimmed in a trapezoid shape with the major (longer) base on the bottom and minor (shorter) base on the top (Figures 3E, 4D-4F and 6). The size of the trapezoid should not exceed the length of the diamond knife (≤ 2 mm).
    4. Mount the resin block on an ultramicrotome holder with the bottom of the sample oriented towards the knife.
    5. Orient the resin block with the sample in the way that the trimmed bottom of the sample (Figure 4A) will be parallel to the glass knife edge (Figure 6).
      Note: When the sample is close to the knife you will be able to see the reflection of the knife on the surface of the resin (this reflection should be parallel).
    6. Cut a few sections using a glass knife.
      Note: The sections can be more than 1 µm thick. The purpose of this point is to see how the sections look like and to orient the specimen properly.
    7. Place a drop of distilled water on a regular or a polylysine-coated slide and put a few sections on that drop.
      Note: Use a wet painting brush (the thinner, the better) to collect the sections.
    8. Dry sections on the slide (see Equipment 30).
    9. Put a drop of toluidine blue stain (Recipe 7) onto the sections for a minute and wash under tap water.
    10. Observe the sections under the light microscope.
      Note: If the sample is oriented in the proper way, you can begin ultra-thin sectioning. If it is not oriented properly (cells are cut askew), adjust the sample position and repeat sectioning (Steps G5-G10).
    11. Mount a diamond knife onto the ultramicrotome and fill the bath with distilled water.
    12. Orient the resin block with the sample in an analogical way as above (see Step G5).
    13. Begin ultrathin sectioning (70 nm thin sections) until you see the ribbon of sections floating on the water.
    14. Using the forceps dip the grid under the water (the dull side with formvar on the top) and collect some sections.
    15. Leave the grids with section on the filter paper or on the round silicon rubber until dry (Figure 7A, numbers 7-9), store in the grid storage box.
      Note: Remember to place the shiny side on the bottom, and dull side with the section on the top.

  8. Immunogold labeling
    Here I describe how to perform the labeling of cell wall epitopes. In this process, primary antibodies bind to specific cell wall epitopes, which are then recognized by secondary antibodies coupled with gold particles for visualization in TEM. The immunolocalization procedure relies on moving the grids over a series of small droplets containing different solutions, antibodies and stain for contrasting, all are placed on the parafilm (Figures 7 and 8). Remember to include controls such as wild type, untreated plants as well as secondary antibody alone, which should not give any labeling.


    Figure 7. Grids for electron microscopy. A. Grids with two sides: a dull side and a shiny side. Grids with the shiny side correspond to numbers 1, 3, 5. Grids with the dull side correspond to numbers 2, 4, 6, 7-9. Sections are mounted on the formvar-coated dull side (numbers 7-9). B. The dull side is in contact with droplets. Usually, the shiny side must be in contact with the filter paper while drying the samples (not shown).


    Figure 8. Immunolocalization procedure. The labeling relies on moving the grids over a series of small droplets containing different solutions (blocking reagent [BR], primary antibodies, PBS buffer, secondary antibody, distilled water) all placed on the parafilm.

    1. Place a piece of parafilm on a clean table.
      Note: Use 70% ethanol to clean the table. The side of parafilm covered by paper is clean and should be on top (where droplets of different solutions will be placed). Avoid touching the parafilm with hands to not contaminate it. Add a few drops of water below the parafilm and stick the corners of the parafilm with a tape to stabilize it and keep flat.
    2. Place a few droplets of BR (depending on the number of grids) in a row on the parafilm (for BR see Recipe 8) (Figure 8A).
    3. Place the grids on droplets of BR and leave it for 15 min at ambient temperature (the dull side of the grid containing the section should be in contact with the droplet) (Figures 7B and 8B).
      Note: Always transfer the grids with clean tweezers. It is recommended to use a set of tweezers (negative-action style). To avoid cross-contamination between antibodies, before touching the grids, always clean tweezers with 70% ethanol, rinse in distilled water and dry with a clean tissue wiper before contact with a grid. Always handle the grids with care. The tweezers should be in contact only with the external ring of the grid.
    4. Put the droplets of primary antibody solutions (Recipe 9) in the second row (use BR instead of primary antibodies for the controls, which will be treated only with secondary antibodies).
    5. Take the grid out of BR droplet, touch the edge of the grid with a clean filter paper (to absorb the reagent) and transfer the grid onto a droplet with primary antibody.
      Note: From the circles of the filter paper, cut small triangles, use the sharp tip to touch the grid edge.
    6. Incubate with primary antibody for 45-60 min at ambient temperature.
      Note: During the incubation in primary and secondary antibodies make sure that the solution will not dry out. To prevent drying, cover the samples with e.g., a large glass Petri dish.
    7. Put droplets of PBS (Recipe 10) in the four next rows.
    8. Take the grid out of the primary antibody droplet, touch the edge of the grid with a clean filter paper (to absorb the reagent), and transfer the grid onto a droplet with PBS.
      Note: Rinse the grids four times in PBS. Leave the grids for 5 min on each droplet. Before transferring to the next PBS droplet, always absorb previous PBS with a filter paper.
    9. Put the droplets of the secondary antibody solution (Recipe 10) into the next row.
    10. Transfer the grids in an analogical way to above (Steps H5 and H8).
    11. Incubate with the corresponding secondary antibody for 60 min at ambient temperature.
    12. Put the droplets of distilled water into the four next rows.
    13. Transfer the grids in an analogical way to above (Steps H5 and H8).
      Note: Rinse the grids four times in distilled water. Leave the grids for 5 min on each droplet. Before transferring to the next water droplet, always absorb previous water with a filter paper.
    14. Take out the grid from the distilled water droplet, touch the edge of the grid with a clean filter paper (to absorb the reagent) and transfer the grid on a filter paper or on a silicon rubber (the dull side with the section should be on top).
    15. Leave the grids for at least 60 min or until dry (cover with a large Petri plate to prevent contamination).
    16. Put a droplet of uranyl acetate (UA) (Recipe 11) on a new piece of the parafilm.
      Note: Briefly spin at maximum speed before use.
    17. Transfer the grid onto a droplet of uranyl acetate (UA) and leave it for 10 min at ambient temperature in darkness (UA is light sensitive).
    18. Take out the grid from UA droplet, touch the edge of the grid with a clean filter paper (to absorb the reagent) and transfer the grid onto the distilled water droplet.
    19. Holding the grids with negative-action style tweezers rinse each grid under tap water for 30 s, then place the grids onto the filter paper or silicon rubber, wait until dry (the dull side with the section should be on the top) (Figure 7A, points 7-9) and store in a grid storage box.
      Note: All the steps involving the use of UA must be carried with special attention by using protective gloves and clothes under the fume hood. All UA waste must be placed in assigned waste bins. Avoid too long incubation in UA as it can give too high contrast.

Recipes

  1. Half strength MS basal medium
    1. Add 2.2 g of MS
    2. Add 10 g of sucrose (1% v/v)
    3. Add distilled water to a final volume of 1 L
    4. Mix the solution on a magnetic stirrer with a magnetic stir bar
    5. Adjust pH to 5.6 by decreasing pH with hydrogen chloride (HCl) or increasing pH with sodium hydroxide (NaOH)
    6. Add 7 g of plant agar (0.7% v/v)
    7. Autoclave the medium
  2. Paraformaldehyde-glutaraldehyde (4% PFA and 0.05% GA) fixative solution
    1. Add 4 g of paraformaldehyde (PFA) powder to a conical flask with 100 ml of 0.1 M phosphate buffer (4% of PFA for total concentration)
    2. Add a magnetic stir bar, cover with aluminum foil to prevent evaporation and place the flask on a magnetic hotplate stirrer (90-100 °C)
    3. Add a few drops of sodium hydroxide solution (NaOH) to speed up dissolving the powder (use 0.1 M concentration of NaOH or lower) and then leave it mixing until the powder dissolves completely (it can take a couple of hours)
    4. Adjust pH to 7.2 by decreasing pH with hydrogen chloride (HCl) or increasing pH with sodium hydroxide (NaOH)
    5. Add 200 μl of 25% glutaraldehyde (GA) (0.05% of GA for total concentration)
    6. Store the fixative solution in a designated (ventilated) refrigerator (for a short storage) or freeze the fixative in -20 °C (for long storage)
    Note: PFA and GA are highly toxic. Conduct all the work under the fume hood, with protective gloves, clothes and mask. Discard the fixative into designated waste. It is recommended to prepare fresh fixative or to use already prepared PFA solution. To not generate additional waste, it is recommended to calculate the exact amount of fixative which is required (see Step A1). To not contaminate the pH meter, it is recommended to use a disposable pH indicator paper instead.
  3. Phosphate buffer (PB), 0.1 M solution (pH = 7.2)
    1. Prepare a stock solution of 0.1 M disodium phosphate (Na2HPO4) and another solution of 0.1 M monosodium phosphate (NaH2PO4) by dissolving these salts in distilled water
    2. Adjust phosphate buffer pH to 7.2 by mixing 68.4 ml of disodium phosphate solution (Na2HPO4) with 31.6 ml of monosodium phosphate solution (NaH2PO4) (Sambrook et al., 1982)
    Note: It is recommended to prepare fresh stock concentrations because salt might precipitate. Confirm the pH with a pH meter measurement. Autoclave phosphate buffer solution before storing in ambient temperature.
  4. Low melting point (LMP) agarose 1% solution
    1. Dissolve 1 g of LMP agarose in 100 ml of distilled water
    2. Warm it up in a microwave until boiling
    3. Wait to cool it down before contact with the plant material
  5. Different ethanol concentrations 10%-95%
    1. Mix ethanol absolute ≥ 99.5% with distilled water to obtain different concentrations
    2. Prepare around 100 ml of each concentration and keep it in closed bottles
  6. Different LRW resin concentrations 10%-75%
    Mix LWR resin with ethanol absolute ≥ 99.5% to obtain different concentrations
  7. Toluidine blue solution
    1. Dissolve 0.5 g of toluidine blue powder in 50 ml of 70% ethanol.
    2. Prepare 1% sodium hydroxide (NaOH) solution by dissolving 0.5 g of NaOH in 50 ml of distilled water
    3. Mix toluidine blue solution with 1% NaOH in ratio 1:5
    4. Adjust pH of NaOH to 2.5 by decreasing pH with hydrogen chloride (HCl) or increasing pH with sodium hydroxide (NaOH)
  8. Blocking Reagent (BR) 1%
    Dissolve 1 g of Bovine Serum Albumin (BSA) in 100 ml of PBS (for 1% of BR for total concentration)
  9. Antibodies solutions
    Primary antibodies are diluted in BR (1:10) and secondary antibodies are diluted in BR (1:50)
  10. Phosphate Buffered Saline (PBS), 0.1 M solution (pH = 7.2)
    8 g sodium chloride (NaCl)
    0.2 g potassium chloride (KCl)
    1.15 g disodium phosphate (Na2PO4)
    0.2 g monopotassium phosphate (KH2PO4)
    Refill with distilled water up to 1 L
    Adjust pH to 7.2 by decreasing it with hydrogen chloride (HCl) or increasing it with sodium hydroxide (NaOH)
    Note: Autoclave phosphate buffer solution before storing in ambient temperature.
  11. Uranyl acetate solution (5%)
    1. Add 0.5 g of UA to 5 ml of double distilled water in a 15 ml tube
    2. Adjust the pH to 3.5 by decreasing it with hydrogen chloride (HCl) or increasing it with sodium hydroxide (NaOH)
    3. Fill the tube with double distilled water up to 10 ml and energetically shake the content
    4. Filter the solution through a filter paper or spin the solution in the centrifuge at maximum speed for 10 min and transfer the liquid part to a new tube
    5. Keep UA solution in the dark
    Note: Prepare UA solution under the fume hood with special attention using protective gloves and clothes. All UA waste must be placed in assigned waste bins. To not contaminate the pH meter, it is recommended to use a disposable pH indicator paper instead.

Acknowledgments

I would like to greatly acknowledge Stéphanie Robert and Richard S. Smith for comments on the manuscript and support of the work. I acknowledge Nicola Trozzi for proofreading the manuscript. I would like to thank Lenore Johansson, Kjell Olofsson and Janusz Kubrakiewicz for sharing their experience. This work was performed at Umeå Core Facility Electron Microscopy at Umeå University. The work was supported by the Swedish Research Council Vetenskapsrådet (grant nos. VR2012-2343 and VR2016-00768), Vinnova (Verket för Innovationssystem) and ERA-CAPS. This protocol was adapted from the published study (Majda et al., 2017).

Competing interests

I declare no conflicts of interest or competing interests.

References

  1. Baskin, T. I. (2005). Anisotropic expansion of the plant cell wall. Annu Rev Cell Dev Biol(21): 203-222.
  2. Cosgrove, D. J. (2005). Growth of the plant cell wall. Nat Rev Mol Cell Biol 6(11): 850-861.
  3. Loqué, D., Scheller, H. V. and Pauly, M. (2015). Engineering of plant cell walls for enhanced biofuel production. Curr Opin Plant Biol 25: 151-161.
  4. Majda, M. (2018). Role of the cell wall in cell shape acquisition. Swedish University of Agricultural Sciences, Umeå, Sweden. Acta Universitatis agriculturae Sueciae. 10: 1652-6880. ISBN 978-9-17-760160-9.
  5. Majda, M. and Robert, S. (2018). The role of auxin in cell wall expansion. Int J Mol Sci 19(4): E951.
  6. Majda, M., Grones, P., Sintorn, I. M., Vain, T., Milani, P., Krupinski, P., Zagorska-Marek, B., Viotti, C., Jonsson, H., Mellerowicz, E. J., Hamant, O. and Robert, S. (2017). Mechanochemical polarization of contiguous cell walls shapes plant pavement cells. Dev Cell 43(3): 290-304 e294.
  7. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1982). Molecular cloning: A laboratory manual. 2nd edition. Cold Spring Harbor Laboratory.
  8. Voiniciuc, C., Pauly, M. and Usadel, B. (2018). Monitoring polysaccharide dynamics in the plant cell wall. Plant Physiol 176(4): 2590-2600. 
  9. Wallace, I. S. and Anderson, C. T. (2012). Small molecule probes for plant cell wall polysaccharide imaging. Front Plant Sci 3: 89.

简介

植物细胞壁由不同的多糖和结构蛋白组成,其形成位于质膜外部的刚性层。该壁也是非常动态的细胞复合物,其特征在于复杂的多糖相互作用和细胞发育过程中的各种修饰。细胞壁成分原位的可视化是非常具有挑战性的,因为细胞壁复合物的尺寸小(纳米尺度),壁多糖的多样性及其复杂的相互作用。该方案描述了用于高分辨率透射电子显微镜(TEM)的不同细胞壁表位的免疫金标记。它提供了植物材料的收集和准备,超薄切片,标本标记和对比的详细程序。优化免疫标记程序工作流程以获得高分辨率TEM的高效碳水化合物标记。该方法用于研究各种植物组织中的植物细胞壁特征,但也可用于植物和动物组织中的其他细胞组分。
【背景】植物生物量主要由细胞壁组成,细胞壁广泛用作日常生活中的能源。植物细胞壁因其转化为生物燃料而备受关注(Loqué et al。,2015; Donev et al。,2018)。在微观尺度上,细胞壁由嵌入复杂基质多糖中的纤维素微纤维组成,例如半纤维素,果胶和结构蛋白。纤维素微纤维是最大的壁聚合物,半径为3-5纳米,长度为数微米(Cosgrove,2005)。纤维素微纤维的取向决定了生长方向和细胞各向异性(Baskin,2005),但纤维素与其他壁组分相互作用,共同改变了壁的性质(在Majda,2018; Majda和Robert,2018中综述)。细胞壁成分的研究历史悠久,可以追溯到不同的化学物质结合到墙体复合材料上时;然而,他们中的许多人有广泛的目标(Wallace和Anderson,2012; Voiniciuc et al。,2018)并且只能通过光学显微镜分辨率观察。相反,免疫金标记的特点是抗体的高特异性和高分辨率成像,可以精确定位壁基质上的壁表位(例如,Majda 等。,2017)。尽管电子显微镜是一种相对较老的方法,但它并未广泛用于植物细胞壁。原因可能是耗时,需要长时间的培训和大量的准备时间。在本协议中,我将引导您完成有关样品制备,所有试剂的特异性和故障排除的所有步骤。

关键字:细胞壁, 细胞壁表位, 多糖, 免疫金标记, 透射电子显微镜

材料和试剂

  1. 附着力滑梯,Polysine,25 x 75 x 1.0 mm(VWR,目录号:631-0107)
  2. 铝箔
  3. 离心管15 ml(PluriSelect,目录号:05-00002-01)
  4. 离心管50 ml(无菌)(PluriSelect,目录号:05-00001-01)
  5. 罐中的压缩空气(例如,空气除尘器PRF 4-44)
  6. 一次性pH指示纸(通用指示纸)(Johnson,目录号:101.3C)
  7. 一次性塑料巴斯德吸管(品牌,目录号:747750)
  8. 双刃剃须刀片(Personna,目录号:171930)
  9. 嵌入胶囊(8毫米扁平,聚丙烯胶囊)(TAAB,目录号:C095)
  10. 滤纸(圆圈,150mmØ),(Whatman,目录号:1001150)
  11. 带塑料按扣帽的玻璃瓶( ca。 41 x 24 mm)(Karl Hecht,目录号:2783/3),或带盖帽的透明玻璃瓶(封闭顶部,PE透明,18毫米,500毫升20 x 40毫米)(VWR,目录号:548-0555)
  12. 用于透射电子显微镜的网格,例如,网格尺寸100目x250μm间距,镍或铜(TAAB,maxtaform HF4,目录号:GM021N; Sigma-Aldrich,目录号:G1528; Agar Scientific,目录号:G2500C)
  13. 轻型3层组织刮水器(VWR,目录号:82003824-CS)
  14. 金属针或解剖针(VWR,目录号:10806-330)
  15. 微量离心管1.5 ml(Sigma-Aldrich,目录号:Z606340)
  16. 显微镜载玻片,25 x 75 x 1.0 mm(VWR,目录号:48300-025或Thermo Scientific,目录号:10144633CF)
  17. 纸标签
  18. Parafilm(Sigma-Aldrich,目录号:P7543)
  19. 铅笔
  20. 移液器吸头
  21. 塑料培养皿(Fisherbrand,目录号:S33580A)
  22. 防护手套(霍尼韦尔,目录号:Dermatril 740)
  23. 圆形硅橡胶(TAAB,目录号:G082)
  24. 带有铝制脊柱的单刃剃须刀片(VWR,目录号:233-0156)
  25. 滑动标签(Agar Scientific)或单面粘性纸
  26. 方形培养皿(120 mm)(Corning,目录号:BP124-05)
  27. 透明胶带(例如,苏格兰威士忌)
  28. 废物容器
  29. 琼脂,植物琼脂(Duchefa Biochemie,目录号:9002-18-0)
  30. 牛血清白蛋白(BSA),冻干粉≥96%(琼脂糖凝胶电泳)(Sigma-Aldrich,目录号:9048-46-8)
  31. 磷酸二钠(Na 2 HPO 4 )(在室温下储存:室温,约21°C)(Sigma-Aldrich,目录号:7558-79-4) )
  32. 蒸馏水
  33. 乙醇实验室试剂,绝对,≥99.5%(易燃,存放在指定地点)(Sigma-Aldrich,目录号:64-17-5)
  34. 甲醛(FA)(10 ml安瓿,16%无甲醇FA)(健康危害,在室温下关闭,或在冷室/冰箱中打开4°C)(Thermo Scientific,目录号:28908)或多聚甲醛(PFA) )用于组织学(CH 2 O) n ,(在冷藏室/冰箱中储存4°C)(JT Baker,目录号:S898-07)
  35. Formvar溶液(1%formvar in dichloroethane,用于显微镜检查)(Sigma-Aldrich,目录号:63148-64-1)
  36. 戊二醛(GA)(I级,1 ml安瓿,25%GA,H 2 O,经过特殊纯化后用作电子显微镜固定剂,线性公式:OHC(CH 2 ) 3 CHO(健康危害,在环境温度下关闭,或在冷室/冰箱中开启4°C)(Sigma-Aldrich,目录号:111-30-8)
  37. 氯化氢(HCl)(Sigma-Aldrich,目录号:7647-01-0)
  38. LR白色树脂中等催化(健康危害,储存在冷藏室/冰箱4°C)(TAAB,目录号:L012)
  39. 磷酸一钾(KH 2 PO 4 )(Sigma-Aldrich,目录号:7778-77-0)
  40. 磷酸单钠(NaH 2 PO 4 )(在环境温度下储存)(Sigma-Aldrich,目录号:7558-80-7)
  41. Murashige和Skoog基础培养基(MS)(Sigma-Aldrich,目录号:M5519)
  42. 氯化钾(KCl)(Sigma-Aldrich,目录号:7447-40-7)
  43. 一抗(PlantProbes: www.plantprobes.net 或佐治亚大学: www.ccrc.uga.edu )
  44. 二抗,例如,EM山羊抗大鼠IgG(H + L):10 nm金(BBI溶液,目录号:014990)或EM山羊抗小鼠IgG(H + L)10 nm Gold(BBI Solutions,目录号:EM.GMHL10)
  45. 氯化钠(NaCl)(Sigma-Aldrich,目录号:7647-14-5)
  46. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:1310-73-2)
  47. 蔗糖(Sigma-Aldrich,目录号:57-50-1)
  48. 用于显微镜的甲苯胺蓝(Sigma-Aldrich,目录号:6586-04-5)
  49. TopVision低熔点琼脂糖(LMP琼脂糖)(在环境温度下保存)(Thermo Scientific,目录号:R0801)
  50. 吐温20(Signa-Aldrich,目录号:P1379)
  51. 醋酸铀酰(UA)(危害健康,在环境温度下储存)(VWR,目录号:541-09-3)
  52. 半强度MS基础培养基(见食谱)
  53. 多聚甲醛 - 戊二醛(4%PFA和0.05%GA)固定液(见食谱)
  54. 磷酸盐缓冲液(PB),0.1 M溶液(pH = 7.2)(见食谱)
  55. 低熔点(LMP)琼脂糖1%溶液(见食谱)
  56. 不同乙醇浓度10%-95%(见食谱)
  57. 不同LRW树脂浓度10%-75%(见食谱)
  58. 甲苯胺蓝溶液(见食谱)
  59. 阻断试剂(BR)1%(见食谱)
  60. 抗体解决方案(见食谱)
  61. 磷酸盐缓冲盐水(PBS),0.1 M溶液(pH = 7.2)(见食谱)
  62. 醋酸铀酰溶液(5%)(见食谱)

设备

  1. 分析平衡
  2. 高压灭菌器(YPO,型号:D66161)
  3. 离心机(Marshall Scientific,Eppendorf,型号:5417C)
  4. 锥形瓶
  5. 干燥器(Thermo Fisher,型号:5311-0250)
  6. 斜口钳例如,斯坦利斜口钳,5(Robosource,目录号:15-11)
  7. 金刚石刀,例如,超45°(硅藻土)
  8. 平烧杯或结晶器(宽直径)
  9. 钳子
  10. 冰箱(-20°C)
  11. 通风柜
  12. 玻璃面包师(50毫升)
  13. 玻璃刀条(Agar Scientific,目录号:AGG336)
  14. 用于组织刀的玻璃刀制造商(LKB,目录号:LKB 7801B,或Agar Scientific,目录号:AGL4158)
  15. 网格存储盒(LKB / Leica目录号:G133,或TAAB Gilder G062)
  16. 实验室烤箱/培养箱(60°C)
  17. 光学显微镜
  18. 带磁力搅拌棒的磁性热板搅拌器
  19. 金属1.5毫升Eppendorf机架
  20. 切片机(Reichart Ultracut)
  21. 微波炉
  22. pH计
  23. 移液器
  24. 防护服和面具
  25. 冰箱(4°C)
  26. 旋转振动器或轨道振动器(IKA KS 130 Basic)
  27. 滑动晾衣架或载玻片染色罐(例如,DWK Life Sciences Wheaton)
  28. 小板钳夹具车间
  29. 薄画笔
  30. 透射电子显微镜(JEOL,型号:JEM-1230)
  31. 镊子:负动作式:薄弯曲尖端(Dumont,目录号:0203-N7-PO)或薄尖端(Dumont,目录号:0302-N0-PO-1)
  32. 镊子:直的日内瓦图案,细尖(Dumont,目录号:0103-0-PO)
  33. 涡旋混合器(Vortex-Genie 2)
  34. 保温板,或滑动干燥电炉(Agar Scientific,型号:AGL4384)或精神灯燃烧器

程序

  1. 植物材料固定
    在本节中,我将描述植物材料制备和固定的程序。该协议是为拟南芥叶子开发的,但它也可以应用于其他生物和组织,如树种的根,芽和木质组织。
    1. 播种前对种子进行消毒
      1. 将少量种子放入1.5ml Eppendorf管中(约为Eppendorf管体积的5%)。
      2. 用吐温20加入1ml 70%乙醇2分钟。
      3. 将70%乙醇和吐温20替换为1ml 95%乙醇1分钟。
      4. 取出乙醇,等待种子干燥。
      注意:在无菌通风橱下进行种子灭菌。
    2. 在室内垂直琼脂平板(配方1)上生长拟南芥幼苗2周,长日照条件(16小时)(温度分别为20°C和18°C)。 br /> 注意:为了使生长同步,将平板放在4°C的冷室/冰箱中2-3天(在黑暗中),将种子春化。
    3. 收获植物材料并将其直接放入装有3-5μl冷多聚甲醛 - 戊二醛(PFA-GA)固定溶液(配方2)的小瓶中。
      注意:记住从每株植物收获相同的叶子数(从底部到顶部计数:子叶,叶1,叶2,叶3,叶4,分生组织)。为了使固定剂能够很好地穿透,切割小方块(~2 mm 2 max)。在叶子的中间部分。收获来自不同植物的至少10片叶子。使用防护手套和衣服在通风橱下进行所有固定步骤(有关处理限制,请参阅零售商提供的安全数据表SDS)。通过使用永久性标记写下名称并在这些标签上粘贴透明胶带来标记样品瓶。
    4. 在环境温度下在干燥器中对样品进行真空吸尘,直到植物碎片下沉(约4小时)。
      注意:可能会发生一些样品在吸尘后漂浮,这表明样品更可能含有氧气。尝试收集位于样品瓶底部的样品。
    5. 将小瓶放在旋转或轨道振荡器上,让样品混合几个小时。
    6. 将样品放在冷藏室/冰箱(4°C)中过夜。
    7. 丢弃PFA-GA固定剂。
    8. 用磷酸盐缓冲液(PB)(配方3)洗涤样品(两次,每次30分钟)。
      注意:在指定的废液瓶中使用一次性塑料巴斯德吸管丢弃PFA-GA固定液和第一次PB洗涤液。加入至少5毫升PB或蒸馏水(PB和蒸馏水越多,洗涤效果越好)进行清洗。在通风橱下,在旋转或轨道振动器上缓慢混合所有洗涤步骤。

  2. 在低熔点(LMP)琼脂糖中包埋植物材料
    在LMP琼脂糖中包埋小植物片段是定位样品进行切片的最佳方法(例如,交叉或纵向)。它标记了芽/根尖的方向(图1A)或远轴和近轴叶侧的定位(图1B)。嵌入还有助于处理和保护样品。


    图1.将植物块置于低熔点(LMP)琼脂糖中。 A-B。样品嵌入长方体形状的琼脂糖块中,呈现一个切割边缘以标记样品的方向(例如,图1A中的左上角)。 A.根部的顶部由切边标记。 B.当切边位于左上方时,叶片的正面位于前面。

    1. 用蒸馏水洗涤样品(两次,每次30分钟)。
    2. 将一层薄薄的(3-5毫米厚)琼脂糖(配方4)倒在方形塑料培养皿上,等待几分钟直至琼脂糖冷却(但不应固化)。
    3. 使用镊子将植物材料放在琼脂糖上(许多块放在同一块板上,但样品之间的距离约为2-3厘米 2 )。
      注意:小心处理植物标本,不要损坏组织。
    4. 加入更多琼脂糖(±3毫米厚的层)覆盖植物标本(等到琼脂糖凝固)(图2A)。
    5. 用嵌入的标本切下琼脂糖的小立方体(一个立方体中的一个标本)(图2B)。
      注意:琼脂糖立方体的大小不应大于嵌入胶囊(8mmØ)的直径。
    6. 切下琼脂糖立方体中的一个角以定向样品(图1,2B和2C)。
    7. 将块移至装有蒸馏水的小瓶中,然后丢弃水,然后再进行下一步。


      图2.将植物材料包埋在琼脂糖中。 A.方形培养皿中填充一层3毫米厚的琼脂糖。 B.小的长方体形状的琼脂糖块,切割的一个边缘(从正面看到所有的叶片)。 C.放大的琼脂立方体,前面的叶子的背面。在这种情况下,大型植物碎片用于可视化,但它们应该小5倍。

  3. 脱水标本包埋在琼脂糖块中
    在这里,我描述了一种从样品中去除水分并使树脂更好渗透的常用方法。时间间隔可以调整;但是,更长的间隔可以提供更好的嵌入质量。理想情况下,脱水过程将在两天内进行,但所有脱水步骤也可在同一天内进行(取决于样品大小)。
    1. 如下所述,将分级乙醇系列中的样品脱水(配方5):
      10%乙醇(两次,每次30分钟)
      20%乙醇(两次,每次30分钟)
      30%乙醇(两次,每次30分钟)
      50%乙醇(两次,每次30分钟)
      注意:加入的乙醇量根据琼脂糖中嵌入的样品的大小和数量而变化。确保琼脂糖块完全被乙醇覆盖。
    2. 将样品置于环境温度(在工作台上)的50%乙醇中过夜。
      注意:用塑料按扣盖关闭小瓶以防止乙醇蒸发。
    3. 继续在分级乙醇系列中对样品进行脱水,如下所示:
      70%乙醇(两次,每次30分钟)
      80%乙醇(两次,每次30分钟)
      90%乙醇(两次,每次30分钟)
      95%乙醇(三次,每次30分钟)
      99.5%乙醇(三次,每次30分钟)
      注意:在旋转或轨道振荡器上进行所有脱水步骤。应使用塑料按扣盖关闭样品瓶。一些中间乙醇稀释液可以省略( 例如 ,20%和80%乙醇),但它可能会影响嵌入质量。对于所有样品瓶,最后一步中的酒精量应相同。

  4. 树脂嵌入
    在该步骤中,用粘性和低溶解性LR白(LRW)树脂代替醇。所有这些步骤必须在轨道振荡器上剧烈混合样品的同时逐步进行。在冷藏室/冰箱(4°C)中执行所有这些步骤,以防止树脂聚合。


    图3.将植物材料嵌入树脂中。 A.嵌入胶囊和纸标签。 B.树脂聚合后的胶囊。 C.切割胶囊以除去树脂块。 D.单独树脂块。 E.修剪树脂块成梯形。

    1. 用已知体积为99.5%乙醇的小瓶缓慢加入几滴树脂,直至树脂含量达到总体积的10%(v / v)。
      注意:所有步骤,包括更换树脂,必须使用防护手套和通风橱下的衣服进行。在冷室/冰箱(4°C)中,将塑料按扣盖在轨道振动器中混合,保持小瓶关闭。务必将树脂丢弃到指定的废物中。
    2. 将小瓶放在轨道振荡器中以将样品混合过夜。
    3. 用以下分级树脂系列中的新树脂交换树脂10%的酒精(配方6):
      10%树脂(约5小时)
      25%树脂(约5小时)
      50%树脂过夜
      75%树脂(约5小时)
      100%树脂过夜
      100%(新鲜)树脂过夜
      注意:可以省略一些中间树脂稀释液( 例如 ,25%和75%),但它可能会影响嵌入质量。 < br />

  5. 在嵌入胶囊和树脂聚合中关闭样品
    这是嵌入程序的最后一步,您必须注意正确定位样品(图1)。琼脂糖块应平放在胶囊底部的中间(图4A和4B),其需要在朝向刀刃的平行方向上取向(参见步骤G,图6)。小心处理胶囊,以防止样品在角落附近滑动,这可能会导致修剪样品和切片时出现一些问题。


    图4.切片前修剪植物标本。 A-B。一片胶囊,其中一片叶子嵌入树脂(A)中。样品应位于胶囊底部的中间(B)。 C-即从底部看到单独的样品树脂:修剪前的树脂(C),修剪方向(D),修整样品(E)。 F.从侧面看到的修剪树脂块。

    1. 将胶囊(图3A)放在架子上,用树脂填充胶囊的一半。
    2. 将样品放入胶囊中,使样品充分定向并用新鲜树脂填充胶囊。
    3. 切割小纸标签,用铅笔标记,然后将它们放在胶囊顶部。
      注意:胶囊应填充树脂,以便通过关闭它,不会留下气泡的空间。使用铅笔标记样品而不是笔或标记,可以用树脂洗掉。
    4. 将胶囊放入培养箱/烘箱(60°C)中24小时(或直至其变为固体)以使树脂聚合。
      注意:切记检查烤箱的温度(60°C)。温度过低会影响聚合反应(树脂不能均匀聚合),而温度过高或温度过长会导致树脂开裂或破裂。
    5. 当树脂变硬时,样品就准备好了(图3B)。
      注意:要知道树脂是否固化,请将金属针压在树脂表面。

  6. 准备用于透射电子显微镜(TEM)的栅格
    用于TEM的样品安装在小网格上,然后将其插入TEM内。网格必须涂有formvar,formvar是保持这些部分的粘合层。确保显微镜载玻片,镊子和实验室玻璃器皿清洁(使用纸巾刮水器和压缩空气清洁表面上的灰尘)。
    1. 添加约。将50ml formvar溶液加入小烧杯中(图5A)。
    2. 将显微镜载玻片垂直放置(保持暗淡的一侧)并大力蘸水。 formvar溶液中一半的载玻片(溶液中不超过约5秒)(图5B)。
      注意:formvar会在侧面形成一层薄膜。
    3. 在干燥架上用垂直位置的薄膜擦干载玻片3-10分钟。
      注意:请勿触摸与formvar解决方案接触的幻灯片的任何部分。
    4. 准备宽直径(≥10cm)的玻璃器皿并用蒸馏水填充。将网格放在圆形硅橡胶或滤纸上(图5C)。
    5. 用锋利的剃刀刀片刮擦滑动边缘,并在滑动件上吹湿空气。
    6. 将载玻片垂直(90°或≥45°)浸入蒸馏水浴中。
      注意:您将看到胶片从玻璃上脱落然后漂浮在水面上。
    7. 使用精细的镊子,将网格放在漂浮的薄膜上,暗淡的一面与formvar接触(尽可能多地放置网格)(图5D和5E)。
      注意:每个网格都有两面:暗边和光泽边(图7A)。切片将安装在formvar涂层的暗淡侧面(此侧应与缓冲液,抗体,蒸馏水,对比污渍接触)(图7B),并且在干燥过程中,有光泽的一面将与滤纸接触。样品(由你决定用formvar涂在哪一面,然后记住要保持一致)。
    8. 准备一张由单面粘性纸或幻灯片标签覆盖的新幻灯片(图5F)。
    9. 抓住垂直位置的滑块并触摸浮动薄膜的一个边缘(图5G)。
    10. 将载玻片浸入水中,这将使带有栅格的薄膜粘附到载玻片上(图5H和5I)。
    11. 保留带有用formvar覆盖的网格的载玻片直到干燥。
      注意:将载玻片存放在密闭的盒子或塑料培养皿中,放在冷藏室/冰箱(4°C)中。


      图5.为TEM准备网格。 A. Baker填充formvar溶液并在载玻片染色罐上滑动。 B.在formvar溶液中浸渍显微镜载玻片。 C.装满蒸馏水的玻璃器皿,带圆形硅橡胶的培养皿和网格,镊子。 D.镊子夹住网格的外圈。 E.在浮动的formvar薄膜上的网格,薄膜的暗淡侧面。 F.将幻灯片标签贴在幻灯片上。 G.在接触浮动薄膜的边缘之前垂直滑动。 H.将载玻片浸入水中,使得带有栅格的薄膜粘附到载玻片上。 I.滑动formvar和网格。

  7. 用于电子显微镜的超薄切片
    准备这些部分最重要的是正确定位样品。记得定期控制部分质量。确保细胞/组织/器官与刀平行定向(图6),如果不是,则校正样品和刀刃之间的角度。


    图6.电子显微镜的超薄切片:首先将样品平行于刀刃定向。当树脂靠近刀时,在树脂表面上会看到刀的反射。这种反射应与样品平行(如果方向适当)。切片进展时,确保细胞/组织/器官与刀平行定向(记住校正样品和刀刃之间的角度)。

    1. 使用小型钳夹车间固定胶囊。
    2. 使用坚硬的单刃剃刀刀片和斜切钳(图3C和3D)去除胶囊。
    3. 用坚硬的单刃剃须刀片修剪样品的底部,然后使用薄的双刃剃须刀片进行更精确的切割(图3E和4C-4F)。
      注意:样品底部与刀刃平行(参见程序E),应修剪成梯形,底部有较大(较长)底座,底部较小(较短)顶部(图3E,4D-4F和6)。梯形的尺寸不应超过金刚石刀的长度(≤2mm)。
    4. 将树脂块安装在超薄切片机支架上,样品底部朝向刀具。
    5. 将树脂块与样品对齐,使样品的修剪底部(图4A)与玻璃刀刃平行(图6)。
      注意:当样品靠近刀子时,您将能够看到刀子在树脂表面上的反射(这种反射应该是平行的)。
    6. 用玻璃刀切几个部分。
      注意:切片厚度可超过1微米。这一点的目的是看看这些部分的外观和正确的方向。
    7. 将一滴蒸馏水放在常规或聚赖氨酸涂层的载玻片上,并在该滴上放几个部分。
      注意:使用湿刷(更薄,更好)来收集部分。
    8. 载玻片上的干燥部分(见设备30)。
    9. 将一滴甲苯胺蓝染色(配方7)放在切片上一分钟,然后在自来水下洗涤。
    10. 观察光学显微镜下的切片。
      注意:如果样品以正确的方式定向,您可以开始超薄切片。如果方向不正确(细胞被歪斜),请调整样品位置并重复切片(步骤G5-G10)。
    11. 将金刚石刀安装到超薄切片机上,并用蒸馏水填充浴槽。
    12. 以与上述类似的方式将树脂块与样品对准(参见步骤G5)。
    13. 开始超薄切片(70纳米薄切片),直到看到漂浮在水面上的切片带。
    14. 使用镊子将网格浸入水下(顶部带有formvar的钝侧)并收集一些部分。
    15. 将网格留在滤纸上或圆形硅橡胶上直至干燥(图7A,编号7-9),存放在网格存储盒中。
      注意:请记住将光亮的一面放在底部,并将光滑的一面放在顶部。

  8. 免疫金标记
    在这里,我描述了如何进行细胞壁表位的标记。在该过程中,一抗与特定细胞壁表位结合,然后通过与金颗粒偶联的二抗识别,以便在TEM中可视化。免疫定位程序依赖于将网格移动到包含不同溶液,抗体和染色的一系列小液滴上以进行对比,所有这些都被放置在封口膜上(图7和8)。请记住包括野生型,未经处理的植物以及单独的二抗等对照,不应给出任何标记。


    图7.用于电子显微镜的网格。 A.具有两面的网格:暗淡的一面和有光泽的一面。具有光泽边的网格对应于数字1,3,5。具有暗淡边的网格对应于数字2,4,6,7-9。切片安装在formvar涂层的暗淡侧面(编号7-9)。 B.暗淡的一面与液滴接触。通常,光泽面必须与滤纸接触,同时干燥样品(未显示)。


    图8.免疫定位程序。标记依赖于将网格移动到包含不同溶液(阻断试剂[BR],一抗,PBS缓冲液,二抗,蒸馏水)的一系列小液滴中在封口膜上。

    1. 将一块封口膜放在干净的桌子上。
      注意:使用70%乙醇清洁桌子。用纸覆盖的封口膜的一面是干净的,应该在顶部(将放置不同溶液的液滴)。避免用手触摸封口膜,以免污染。在封口膜下面加几滴水,用胶带粘在封口膜的角落,使其稳定并保持平整。
    2. 在封口膜上连续放置几滴BR(取决于网格的数量)(对于BR,参见配方8)(图8A)。
    3. 将网格放置在BR的液滴上,并在环境温度下放置15分钟(包含该部分的网格的暗淡侧面应与液滴接触)(图7B和8B)。
      注意:务必使用干净的镊子转移网格。建议使用一套镊子(负面动作式)。为避免抗体之间的交叉污染,请始终用70%乙醇清洁镊子,用蒸馏水冲洗并用干净的纸巾擦拭干净,然后再与网格接触。务必小心处理网格。镊子应仅与电网的外环接触。
    4. 将第一抗体溶液(配方9)的液滴放入第二行(使用BR代替一抗用于对照,其仅用二抗处理)。
    5. 将网格从BR液滴中取出,用干净的滤纸接触网格边缘(以吸收试剂)并将网格转移到带有一抗的液滴上。
      注意:从滤纸的圆圈中剪切小三角形,使用尖锐的笔尖触摸网格边缘。
    6. 在室温下与一抗孵育45-60分钟。
      注意:在一抗和二抗孵育期间,确保溶液不会变干。为防止干燥,请使用 覆盖样品, ,一个大玻璃培养皿。
    7. 将PBS液滴(配方10)放入下一行。
    8. 将网格从一级抗体液滴中取出,用干净的滤纸(以吸收试剂)接触网格边缘,然后将网格转移到含有PBS的液滴上。
      注意:用PBS冲洗网格四次。在每个液滴上留下网格5分钟。在转移到下一个PBS液滴之前,始终用滤纸吸收之前的PBS。
    9. 将二抗溶液(配方10)的液滴放入下一行。
    10. 以类似的方式将网格传输到上面(步骤H5和H8)。
    11. 在环境温度下与相应的二抗孵育60分钟。
    12. 将蒸馏水滴放入下一排。
    13. 以类似的方式将网格传输到上面(步骤H5和H8)。
      注意:用蒸馏水冲洗网格四次。在每个液滴上留下网格5分钟。在转移到下一个水滴之前,请务必使用滤纸吸收以前的水。
    14. 从蒸馏水滴中取出网格,用干净的滤纸(以吸收试剂)接触网格的边缘,然后将网格转移到滤纸或硅橡胶上(暗淡的一侧应该打开部分)最佳)。
    15. 将网格放置至少60分钟或直至干燥(用大的培养皿覆盖以防止污染)。
    16. 将一滴乙酸双氧铀(UA)(食谱11)放在一块新的封口膜上。
      注意:以最大速度短暂旋转。
    17. 将网格转移到乙酸铀酰(UA)液滴上,在环境温度下在黑暗中放置10分钟(UA对光敏感)。
    18. 从UA液滴中取出网格,用干净的滤纸(以吸收试剂)接触网格边缘,并将网格转移到蒸馏水滴上。
    19. 用负片式镊子夹住网格,用自来水冲洗每个网格30秒,然后将网格放在滤纸或硅橡胶上,等待干燥(带有截面的暗淡侧面应位于顶部)(图7A) ,点7-9)并存储在网格存储盒中。
      注意:所有涉及使用UA的步骤必须在通风橱下使用防护手套和衣服时特别注意。所有UA废物必须放在指定的废物箱中。避免在UA中孵育太长时间,因为它会产生过高的对比度。

食谱

  1. 半强度MS基础培养基
    1. 加入2.2克MS
    2. 加入10克蔗糖(1%v / v)
    3. 加入蒸馏水至最终体积为1升
    4. 在磁力搅拌器上用磁力搅拌棒混合溶液
    5. 通过用氯化氢(HCl)降低pH或用氢氧化钠(NaOH)增加pH将pH调节至5.6
    6. 加入7克植物琼脂(0.7%v / v)
    7. 高压灭菌培养基
  2. 多聚甲醛 - 戊二醛(4%PFA和0.05%GA)固定溶液
    1. 将4 g多聚甲醛(PFA)粉末加入带有100 ml 0.1 M磷酸盐缓冲液(4%PFA总浓度)的锥形瓶中
    2. 添加磁力搅拌棒,盖上铝箔以防止蒸发,并将烧瓶放在磁性热板搅拌器上(90-100°C)
    3. 加入几滴氢氧化钠溶液(NaOH)以加速溶解粉末(使用0.1 M或更低浓度的NaOH),然后将其混合直至粉末完全溶解(可能需要几个小时)
    4. 通过用氯化氢(HCl)降低pH或用氢氧化钠(NaOH)增加pH将pH调节至7.2
    5. 加入200μl25%戊二醛(GA)(总浓度为0.05%GA)
    6. 将固定液储存在指定(通风)冰箱中(短期储存)或将固定液冷冻在-20°C(长期储存)
    注意:PFA和GA具有高毒性。在通风橱下进行所有工作,带防护手套,衣服和面罩。将固定剂丢弃到指定的废物中。建议准备新鲜的固定剂或使用已经制备的PFA溶液。为了不产生额外的浪费,建议计算所需的固定剂的确切量(参见步骤A1)。为了不污染pH计,建议使用一次性pH指示纸。
  3. 磷酸盐缓冲液(PB),0.1 M溶液(pH = 7.2)
    1. 制备0.1M磷酸二钠(Na 2 HPO 4 )和另一种0.1M磷酸二氢钠溶液(NaH 2 PO 的储备溶液> 4 )将这些盐溶解在蒸馏水中
    2. 通过将68.4 ml磷酸二钠溶液(Na 2 HPO 4 )与31.6 ml磷酸二氢钠溶液(NaH 2 PO 4 )(Sambrook et al。,1982)
    注意:建议准备新鲜浓度,因为盐可能会沉淀。用pH计测量确认pH。在环境温度下储存前,高压灭菌磷酸盐缓冲溶液。
  4. 低熔点(LMP)琼脂糖1%溶液
    1. 将1g LMP琼脂糖溶于100ml蒸馏水中
    2. 用微波炉加热至沸腾
    3. 在接触植物材料之前,请等待冷却
  5. 不同乙醇浓度10%-95%
    1. 用蒸馏水将乙醇绝对浓度≥99.5%混合,得到不同浓度
    2. 准备每种浓度约100毫升,并将其保存在密闭瓶中
  6. 不同的LRW树脂浓度为10%-75%
    将LWR树脂与乙醇绝对≥99.5%混合,得到不同浓度
  7. 甲苯胺蓝溶液
    1. 将0.5g甲苯胺蓝粉末溶于50ml 70%乙醇中。
    2. 通过将0.5g NaOH溶解在50ml蒸馏水中制备1%氢氧化钠(NaOH)溶液
    3. 将甲苯胺蓝溶液与1%NaOH以1:5的比例混合
    4. 通过用氯化氢(HCl)降低pH或用氢氧化钠(NaOH)增加pH将NaOH的pH调节至2.5
  8. 阻断试剂(BR)1%
    将1克牛血清白蛋白(BSA)溶于100毫升PBS中(总浓度为1%BR)
  9. 抗体解决方案
    一抗在BR(1:10)中稀释,二抗在BR中稀释(1:50)
  10. 磷酸盐缓冲盐水(PBS),0.1 M溶液(pH = 7.2)
    8克氯化钠(NaCl)
    0.2克氯化钾(KCl)
    1.15g磷酸二钠(Na 2 PO 4 )
    0.2克磷酸二氢钾(KH 2 PO 4 )
    重新加注蒸馏水至1升
    通过用氯化氢(HCl)将其降低或用氢氧化钠(NaOH)将其增加来将pH调节至7.2 注意:在环境温度下储存前,先将高压灭菌磷酸盐缓冲溶液。
  11. 醋酸铀酰溶液(5%)
    1. 在15ml管中加入0.5g UA至5ml双蒸水中
    2. 通过用氯化氢(HCl)降低pH值或用氢氧化钠(NaOH)将pH值调节至3.5来调节pH值
    3. 用高达10毫升的双蒸水填充试管并大力摇动内容物
    4. 通过滤纸过滤溶液或在离心机中以最大速度旋转溶液10分钟并将液体部分转移到新管中
    5. 让UA解决方案在黑暗中
    注意:在通风橱下准备UA溶液,并特别注意使用防护手套和衣服。所有UA废物必须放在指定的废物箱中。为了不污染pH计,建议使用一次性pH指示纸。

致谢

我想非常感谢StéphanieRobert和Richard S. Smith对手稿的撰写和对工作的支持。我承认Nicola Trozzi校对手稿。我要感谢Lenore Johansson,Kjell Olofsson和Janusz Kubrakiewicz分享他们的经验。这项工作在Umeå大学的UmeåCoreFacility电子显微镜进行。这项工作得到了瑞典研究委员会Vetenskapsrådet(授权号VR2012-2343和VR2016-00768)和Vinnova(VerketförInnovationssystem)和ERA-CAPS的支持。该方案改编自已发表的研究(Majda et al。,2017)。

利益争夺

我声明任何利益冲突或竞争利益。

参考

  1. Baskin,T。I.(2005)。 植物细胞壁的各向异性扩增。 Annu Rev Cell Dev Biol (21):203-222。
  2. Cosgrove,D.J。(2005)。 植物细胞壁的生长。 Nat Rev Mol Cell Biol 6(11):850-861。
  3. Donev,E.,Gandla,M.L。,Jonsson,L。J.和Mellerowicz,E.J。(2018)。 为生物精炼厂设计木材非纤维素多糖。 Front Plant Sci 9:1537。
  4. Loqué,D.,Scheller,H。V.和Pauly,M。(2015)。 加强生物燃料生产的植物细胞壁工程。 Curr Opin Plant Biol 25:151-161。
  5. Majda,M。(2018年)。 细胞壁在细胞形状获取中的作用。瑞典农业科学大学,瑞典于默奥。 Acta Universitatis agriculturae Sueciae。 10:1652-6880。 ISBN 978-9-17-760160-9。
  6. Majda,M。和Robert,S。(2018)。 生长素在细胞壁扩增中的作用。 Int J Mol Sci 19(4):E951。
  7. Majda,M.,Grones,P.,Sintorn,IM,Vain,T.,Milani,P.,Krupinski,P.,Zagorska-Marek,B.,Viotti,C.,Jonsson,H.,Mellerowicz,EJ, Hamant,O。和Robert,S。(2017)。 连续细胞壁的机械化学极化形成植物路面细胞。 Dev Cell 43(3):290-304 e294。
  8. Sambrook,J.,Fritsch,E.F。和Maniatis,T。(1982)。分子克隆:实验手册。第2版。冷泉港实验室。
  9. Voiniciuc,C.,Pauly,M。和Usadel,B。(2018)。 监测植物细胞壁中的多糖动态。 植物生理学 176(4):2590-2600。&nbsp;
  10. Wallace,I。S.和Anderson,C。T.(2012)。 用于植物细胞壁多糖成像的小分子探针。 Front Plant Sci 3:89。
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引用:Majda, M. (2019). Visualization of Plant Cell Wall Epitopes Using Immunogold Labeling for Electron Microscopy. Bio-protocol 9(7): e3205. DOI: 10.21769/BioProtoc.3205.
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