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Feb 2020

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Multitarget Immunohistochemistry for Confocal and Super-resolution Imaging of Plant Cell Wall Polysaccharides
植物细胞壁多糖共聚焦和超分辨成像的多靶点免疫组织化学研究   

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Abstract

The plant cell wall (PCW) is a pecto-cellulosic extracellular matrix that envelopes the plant cell. By integrating extra-and intra-cellular cues, PCW mediates a plethora of essential physiological functions. Notably, it permits controlled and oriented tissue growth by tuning its local mechano-chemical properties. To refine our knowledge of these essential properties of PCW, we need an appropriate tool for the accurate observation of the native (in muro) structure of the cell wall components. The label-free techniques, such as AFM, EM, FTIR, and Raman microscopy, are used; however, they either do not have the chemical or spatial resolution. Immunolabeling with electron microscopy allows observation of the cell wall nanostructure, however, it is mostly limited to single and, less frequently, multiple labeling. Immunohistochemistry (IHC) is a versatile tool to analyze the distribution and localization of multiple biomolecules in the tissue. The subcellular resolution of chemical changes in the cell wall component can be observed with standard diffraction-limited optical microscopy. Furthermore, novel chemical imaging tools such as multicolor 3D dSTORM (Three-dimensional, direct Stochastic Optical Reconstruction Microscopy) nanoscopy makes it possible to resolve the native structure of the cell wall polymers with nanometer precision and in three dimensions.

Here we present a protocol for preparing multi-target immunostaining of the PCW components taking as example Arabidopsis thaliana, Star fruit (Averrhoa carambola), and Maize thin tissue sections. This protocol is compatible with the standard confocal microscope, dSTORM nanoscope, and can also be implemented for other optical nanoscopy such as STED (Stimulated Emission Depletion Microscopy). The protocol can be adapted for any other subcellular compartments, plasma membrane, cytoplasmic, and intracellular organelles.

Keywords: Plant cell wall (植物细胞壁), Immunohistochemistry (免疫组织化学), Cell wall polysaccharides (细胞壁多糖), Super-resolution microscopy (超分辨显微镜), 3D dSTORM nanoscopy (3D dSTORM纳米显微技术), Morphogenesis (形态发生)

Background

PCW is an intricate material, which is durable but also undergoes constant structural changes in response to internal and external stimuli such as tissue expansion or pathogen attack. Nevertheless, how these seemingly contradictory features, mechanical strength and structural adaptation, cooperate, remains an unresolved question in plant cell biology. PCW contains cellulose, hemicellulose, different variants of pectin and various proteins, the architecture of which is highly organized (Peaucelle, 2018). The pectin family is composed of several polymers. The most abundant, homogalacturonan, can be demethylated after cell wall insertion. This change in chemistry is a significant step in the process of cell elongation, differentiation, and directional growth (Peaucelle et al., 2012). Several lines of evidence suggest that morphogenesis and cell differentiation are dependent on local changes in cell wall chemistry and cell wall polymer organization (Yang et al., 2016; Anane et al., 2017; Zhao et al., 2019; Haas et al., 2020). Therefore, detailed knowledge of PCW components architecture is essential for understanding plant growth. Historically, the cell wall structure has been studied using biochemical methods that involve disintegrating the tissue and destroying the native organization of its polymers (Höfte and Voxeur, 2017). Other imaging modalities, such as electron microscopy (EM) (Anane et al., 2017) and atomic force microscopy (AFM) (Zhang et al., 2017) are used for in situ cell wall observation, but these techniques often lack chemical contrast, providing only correlative quantification. Multicolor immunohistochemistry (IHC) can reveal multiple targets within a tissue section, and their spatial organization resolved in three dimensions. Despite its widespread use in the cell biology field, multicolor IHC is not yet a standard tool for studies of the cell wall. A broad palette of antibodies and probes against cell wall targets, coupled with multicolor IHC, together with the high resolving power of dSTORM (< 40 nm), permits quantitative in situ chemical analysis of cell wall nanostructure. Other imaging techniques for cell wall analysis on tissue cuts exist, such as Raman (Wightman et al., 2019) and FTIR (Mravec et al., 2017; Cuello et al., 2020). These techniques are based on the characteristic absorption/transmission of different chemical components and can measure some changes in cell wall composition at the cellular level but can lack sensitivity, and their ability to observe changes at the subcellular level is severely limited.

The dSTORM permits localization of biomolecules with the precision of 5-10 nm; however, the final dSTORM resolution, typically around 40 nm, is limited by the size of the antibody complex (~15-30 nm). The immunogold Electron Microscope (iEM) is also used in combination with ICH to study cellular structures at high resolutions. IEM is comparable to dSTORM resolution and is also limited by the antibody complex size. The size of the nanogold particle defines iEM contrast and resolution; > 1 nm gold nanoparticles are available; however, such a small particle limits the contrast, and larger particles are often used. IEM, contrasted with dSTORM, has several additional drawbacks: (1) the primary antibodies probed with protein-A (or G) gold complexes do not penetrate through the resin-embedded sample, and only recognize the surface epitopes, although the serial and ultra-thin cryo-sectioning technique can resolve this problem (50 nm, -120 °C [Slot, 1989]); (2) samples are mostly single-labeled; yet by using different nanoparticle size, two or three epitopes can be tagged, but it requires the technical experiences of ultra-thin cryo-sectioning technique (Slot and Geuze, 2007); (3) IEM has low labeling and detection efficiency (3-5 orders of magnitude less than dSTORM (Majda et al., 2017; Haas et al., 2020), which is also related to the fact, that only the surface epitopes are labeled. Multicolor 3D dSTORM nanoscopy can, therefore, provide unprecedented insights into the nanoarchitecture of the native-structure of the cell wall polysaccharides, beyond the applicability of the aforementioned techniques. DSTORM permits a quantitative three-dimensional (3D) nanoimaging of cells and tissues (Heilemann et al., 2008; Huang et al., 2008; Van De Linde et al., 2011; Sydor et al., 2015; Xu et al., 2018). Applied mainly to cellular systems, it unveiled new structural organizations of proteins, e.g., synaptic nanodomains, trans-synaptic nanocolumns, DNA recombinase nanofilaments, nuclear envelope pore structure, cytoskeleton, mitochondria, adhesion complexes, and chromatin transcriptional landscape (Shroff et al., 2008; Shim et al., 2012; Löschberger et al., 2012; Jakobs and Wurm, 2014; Prakash et al., 2015; Boettiger et al., 2016; Sellés et al., 2017; Dlasková et al., 2018; Haas et al., 2018a and 2018b; Pan et al., 2018; Xia et al., 2019; Chen et al., 2020; Wäldchen et al., 2020). Yet, due to the limitation of single-cell model systems requiring tissue-level imaging, its utilization in plant science is almost absent (Liesche et al., 2013; Komis et al., 2015; Haas et al., 2020). Here we present a detailed protocol for the sample preparation compatible with a standard confocal microscope and a dSTORM nanoscope on thin plant tissue sections.

Materials and Reagents

  1. 8-well ibidi µ-slides for dSTORM sample preparation and imaging (Ibidi, catalog number: 80827 )
  2. Biopsy cassette 42 x 28 x 6 mm (Leica Biosystems, ID: IP-Biopsy-cassette-III )
  3. Metal base mold (Leica Biosystems, ID: metal-base-molds )
  4. Charged slides by poly-L-lysine treatment for confocal imaging sample preparation and imaging such as ProbeOn Plus (Fisher Scientific catalog number: 22-230-900 or PolysineTM Microscope Adhesion Slides (Thermo Scientific catalog number: J2800AMNZ)
  5. Aluminum foil (you can get it from a local store)
  6. Coverglass, rectangular, 24 x 60 mm, #1.5 thickness (Knittel Glass, catalog number: 425-2460 )
  7. Eppendorf Safe-lock Microcentrifuge Tubes, 1.5 ml or 2 ml volume (Eppendorf, catalog numbers: 022-36-320-4 [1.5 ml] , 022-36-335-2 [2 ml])
  8. Cardboard Slide Tray (Heathrow Scientific, catalog number: HD9902 )
  9. Paper towels (Divers/Dutcher, catalog number: 475040 )
  10. Empty pipette tips box for homemade humidified chamber for ibidi µ-slides, such as Adamas-Beta 1,000 µl volume tips
  11. Nitrile gloves, such as FisherbrandTM Comfort Nitrile Gloves (Fisher Scientific, catalog number: 15642367)
  12. National DiagnosticsTM HistoclearTM Tissue clearing agent (Fisher Scientific, HS-200-1GAL, CAS number: 5989-27-5)
  13. Embedding medium paraffin (Leica Biosystems, ID: em-400-embedding-medium-paraffin)
  14. Oxoid Skim Milk Powder (Thermo Fisher Scientific, catalog number: LP0031B )
  15. CMB3a Crystalline cellulose-binding module, his-tagged and recombinant CBM protein (PlantProbes, catalog number: CMB3a ) (Blake et al., 2006; Hernandez-Gomez et al., 2015), store at -20 °C
  16. Mouse monoclonal antibody against low esterified homogalacturonan (degrees of methyl-esterification (DM) up to 40%) 2F4 (PlantProbes, catalog number: 2F4 ) (Liners, Thibault and Van Cutsem, 1992), store at 4 °C
  17. Rat IgM monoclonal antibody against high-esterified homogalacturonan LM20 (PlantProbes, catalog number: LM20 ) (Verhertbruggen et al., 2009), store at 4 °C
  18. Rat IgA monoclonal antibody against partially methyl-esterified epitopes of homogalacturonan (PlantProbes, catalog number: JIM7 ) (Knox et al., 1990), store at 4 °C
  19. Rat IgG2a monoclonal antibody against xyloglucan binding preferentially to the XLLG motif of xyloglucan LM24 (PlantProbes, catalog number: LM24 ) (Tanackovic et al., 2016), store at 4 °C
  20. Anti-His tag polyclonal antibody produced in chicken, Anti-6X-His tag (Abcam, catalog number: ab9107 ) store at -20 °C
  21. Anti-His tag polyclonal antibody produced in rabbit (Merck, catalog number: SAB4301134 ), store at -20 °C
  22. Rabbit PDM antibody against Mannan was a kind gift by Paul Dupree (Handford et al., 2003; Yang et al., 2016), store at 4 °C. For availability, please contact raymond.wightman@slcu.cam.ac.uk
  23. Recombinant and His-tagged CBM4 of Cellulomonas fimi (ATCC 484) endoglucanase C (CBD4N1) produced from E. coli. CBM4 was a kind gift by Harry Gilbert (Johnson et al., 1996; Blake et al., 2006), store at 4 °C. For availability, please contact raymond.wightman@slcu.cam.ac.uk
  24. Goat anti-rat CF568 secondary antibody (Sigma, catalog number: SAB4600086 ), store at -20 °C
  25. Goat anti-mouse CF568 conjugated F(ab)’2 secondary antibody fragment (Sigma, catalog number: SAB4600400) , store at -20 °C
  26. Goat anti-mouse F(ab')2 secondary antibody fragment conjugated to Alexa Fluor 647 (Stratech Scientific, catalog number: 115-607-003-JIR) , store at 4 °C
  27. Donkey anti-mouse F(ab')2 secondary antibody fragment conjugated to Alexa Fluor 647 (Abcam, catalog number: ab181292) , store at 4 °C
  28. Donkey anti-Chicken F(ab’)2 secondary antibody fragment conjugated to Alexa Fluor 647 (Jackson immunoresearch, catalog number: 703-606-155) , store at 4 °C
  29. Goat anti-mouse F(ab')2 secondary antibody fragment conjugated to ATTO488 (Hypermole, catalog number: 2402-0.5MG) , store at -20 °C
  30. Goat anti-mouse Alexa 488 conjugated secondary antibody (Sigma, catalog number: SAB4600387) , store at 4 °C
  31. Donkey anti-rat F(ab')2 secondary antibody fragment conjugated to Alexa Fluor 647 (Abcam, catalog number: ab150151) , store at 4 °C
  32. ProLong Gold Antifade Mountant (Thermo Ficher Scientific, catalog number: P36934 ), store at 4 °C
  33. Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: 254134 ), store at RT
  34. Poly-L-lysinesolution 0.1% (w/v) in H2O (Merck, catalog number: P8920 ), store at RT
  35. Ethanol absolute anhydrous (CARLO ERBA reagents, catalog number: 4146082 ), store at RT
  36. Acetic Acid ≥ 96% (AnalaR NORMAPUR, catalog number: 20099.324 ), store at RT
  37. Formaldehyde (Sigma-Aldrich catalog number: F1635 ), store at RT
  38. Arabidopsis inflorescence meristem fixed at 1 cm length stage, and cotyledon of 3 days-old seedling, leaf rachis of star fruit (Supplemental Figure 1AG), immature maize leaf blade at 6th node (Supplemental Figure 1AF)
  39. Nail polish (you can get it from a local drugstore)
  40. Pectolyase for enzymatic extraction of pectins (Sigma, catalog number: P3026 or Magazyme,catalog number: E-PLYCJ )
  41. Calcofluor White Stain for general cell wall staining (Sigma Aldrich, catalog number: 18909-100ML-F )
  42. 2F4 Buffer (T/Ca/S buffer final concentration) (see Recipes), store at RT
  43. FAA solution (see Recipes), store at RT
  44. Formaldehyde diluted in 10x 2F4 buffer (see Recipes)
  45. Citrate-Phosphate Buffer for Pectolyase incubation (pH 4.8) (see Recipes)

Equipment

  1. Dispensable microtome knife (Microm Microtech, catalog number: F/MM35p )
  2. Micro tweezer (Ideal-tek, catalog number: 5-SA )
  3. Glass Rectangular 250 ml Coplin Staining Jar, with Lid (Wheaton, catalog number: 900620 )
  4. FisherbrandTM Microscope Slide Box for homemade humidified chamber for Polysine microscope slides (Fisher Scientific, catalog number: 22363400
  5. Brush for the capture of serial cuts obtained by the microtome (Supplemental Figure 1K, any fine brush from local stationery store, such as Etude, P10531.00 , #2)
  6. HistoCore Arcadia Heated Paraffin Embedding Station (Leica, ID: 14039357258 )
  7. Leica EG F Electric Heatable Forceps
  8. Microtome (Leica, model: RM2265 )
    For more information, see user’s manual.
  9. Fridge 4 °C
  10. Incubation chamber, such as Selecta (set at 60 °C), airflow not necessary
  11. Laboratory freezer (-20 °C), such as Kirsch FROSTER LABO 330 ULTIMATE
  12. Electronic laboratory heating plate (BIO-OPTICA Milano SpA, catalog number: 40-300-300 )
  13. Laboratory chemical fume hood

For the imaging steps:
  1. Any Fluorescence Microscope equipped with four laser lines for the detection in UV (405 nm), green (488 nm), red (561 nm) and far red (633 nm) (here we use Zeiss LSM 710 , Zeiss Oberkochen Germany)
    Brochure available here.
    Specification
    1. Stands: Inverted (Axio Observer Z1 with side port port)
    2. Z drive and XY stage (option): Motorized XY-scanning stage, with Mark & Find function (xyz) and Tile Scan (mosaic scan); the smallest increments 1 μm
    3. Objectives: x10, x25, x40, x63, x100
    4. Lasers: Argon laser (458, 488, 514 nm), HeNe laser (633 nm), diode laser 405 nm, and DPSS laser 561 nm
      1. 405 nm for Calcofluor White
      2. 488 nm for Alexa 488 and ATTO 488
      3. 561 nm for CF568 and Alexa 568
      4. 633 nm for Alexa 647
    5. Scanning Module
      Model: Scanning module with 32 spectral detection channels (QUASAR)
      Scanners: Two independent, galvanometric scan mirrors with ultra-short line and frame fly back.
      Scanning resolution: 4 x 1 to 6,144 x 6,144 pixels
      Scanning speed: 8 frames/sec with 512 x 512 pixels.
      Number of fluorescence-spectral detectors: 2
      Bright field transmission detector: Installed
    6. Software
      Standard Software: ZEN2010
      Optional Softwar: Image J or Fiji
      Computer specification: HP Z800 Workstation, 64-bit Windows 7 Ultimate 2009, 24 Gb RAM, Intel® Xeon® CPU, X5650, Two processors 2.67 GHz, 2.66 GHz
      Wild-field microscope (Nikon N-STORM)

Software

  1. Grafeo (Custom made software for dSTORM data analysis and visualization, https://github.com/inatamara/Grafeo-dSTORM-analysis- (Haas et al., 2018b)
  2. Fiji (https://imagej.net/Fiji/Downloads)

Procedure

In this protocol, we use Arabidopsis thaliana cotyledons meristem, star fruit (Averrhoa carambola) rachis (central fiber in the compound leaf), and maize leaf samples.

  1. Sample Preparation
    1. Star fruit was grown in soil in the greenhouse for two years, and leaf rachis was harvested at the begging of April 2019. Maize was grown in soil in the field sown in May and harvested at the beginning of July 2019 before its flowering. Arabidopsis was grown on MS (Murashige and Skoog) solid nutrient agar medium without sucrose, in constant light at 21 °C, and seedlings were harvested at three days after germinations (3 DAG) (Peaucelle, Wightman and Höfte, 2015). Arabidopsis meristem was harvested from a plant grown on soil in the growing chamber and harvested when the inflorescence was 1 cm long; flowers with visible sepals were removed, keeping all the closest flower buds ( as described in Yang et al., 2016)
    2. Leaf rachis of star fruit was cut with a dispensable microtome knife into 0.5 cm-length-explant (Supplemental Figures 1AG, 2A). The immature region of the Maize shoot tip was isolated from the plantlet (Supplemental Figure 1AF). Maize immature leaf-blade without midrib at 6th node was cut into 5 mm x 5 mm square (Supplemental Figure 2C).
    3. A volume range to fix different organs of Arabidopsis is typically between 1:10 to 1:100 (tissue to fixative volume), here we placed 20 seedlings without the excision of cotyledon in 1.5 ml Eppendorf tube filled with a 1.0 ml of FAA solution. The volume ratio for maize leaf explants was 1:3, and the volume ratio of star fruit leaf rachis explants was 1:100. We placed 5 explants of maize leaves and 10 explants of star fruit leaf rachis in 2.0 ml Eppendorf tube filled with 1.5 ml of FAA solution, respectively. The significant protocol steps are shown in the Supplemental Figure 1, and the preparation of tissue explants, and the microtome cutting position in Supplemental Figure 2.

  2. Fixation and sample embedding
    1. Wear nitrile gloves.
    2. Fix plant organs in the FAA solution in the 1.5/2 ml Eppendorf tube for 1 h at room temperature or overnight at 4 °C. Store fixed samples at 4 °C for up to 1 month in 70% EtOH (see Notes). No vacuum treatment is necessary.
    3. Dehydrate the samples by incubating at room temperature in successive ethanol dilutions for at least 30 min each: 70%, 95%, and twice 100% ethanol. Use approximately 1.5 ml volume in the Eppendorf tube.
    4. Replace ethanol with 1.5 ml of 50% Histoclear in ethanol in Eppendorf for 1 h at room temperature, followed by 1.5 ml of 100% Histoclear for 1 h each at room temperature (this time depends on the thickness of the sample. Reduce the incubation time for very thin tissue cuts, such as roots or Hypocotyl to 30 min).
    5. Transfer the sample to a biopsy cassette (IP-Biopsy-Cassette-III, Leica Biosystems). Start to melt the paraffin in an incubation chamber preheated to 60-70 °C, 12 h prior to Procedure B. Completely melted paraffin and new Histoclear should be preheated to 60-70 °C prior to Step B6 to make 50% paraffin in Histoclear. During a typical experiment, we use approximately 600 ml of paraffin and 100 ml Histoclear in Step B6.
      Preheat the 250 ml glass jar containing 200 ml of the final of 50% Histoclear and 50% paraffin before Step B6.
      Note: We recommend using the EM-400 Embedding Medium Paraffin, which has a low melting point (56-57 °C). This helps the precise positioning of the sample and reduces tissue distortion.
    6. Replace the Histoclear with the paraffin in the incubation chamber by immersing biopsy cassettes in a 250 ml Coplin glass staining jar (Supplemental Figure 1B). Start with a mixture of 50% Histoclear and 50% paraffin in 200 ml for 3 h at 60-70 °C, followed by twice 200 ml of 100 % paraffin for 3 h, and finally 200 ml of 100% paraffin overnight at 60-70 °C.
    7. Turn on the HistoCore Arcadia H Heated Paraffin Embedding Station (Supplemental Figure 1C) and set the HistoCore Arcadia H in operation mode before Step B8 (Set the temperatures of paraffin tank, dispenser, working surface at ~70 °C and a cold spot at 4 °C. For more information, See the HistoCore Arcadia H user manual).
    8. Take out biopsy cassettes and hot 100% paraffin in 250 ml Coplin glass staining jar from the incubator. Let biopsy cassettes floating in melted paraffin of heated instrument tray on a hot preparation surface set at ~70 °C of HistoCore Arcadia H before the solidifying the paraffin at room temperature (Supplemental Figure 1D).
    9. Transfer of the samples from the biopsy cassette to the metal base mold using electric heatable forceps (Supplemental Figure 1E). Samples should be positioned perpendicular to the cutting plane before solidifying the paraffin at room temperature (Supplemental Figures 1F to 1H and 2D).
    10. Once positioned, rapid solidification of the paraffin could be achieved using a cold spot (small round metallic plate shown in Supplemental Figure 1G), usually at 4 °C.
      Note: Rapid cooling of the paraffin will limit the formation of paraffin crystals, and make the preparation transparent. In contrast, slow cooling will lead to crystal formation and the widening of the preparation. The crystallized paraffin is slightly more rigid and limits greatly the ability to localize the sample during microtome cutting.
    11. After Step B10, store the samples at 4 °C overnight. Before cutting samples with the microtome, keep the room temperature bellow 22 °C to prevent the paraffin softening.
    12. Turn on the microtome. We recommend using the microtome in the manual mode. The use of the motorized mode is possible, but we did not test it. The settings for the cut thickness are: 3-5 µm (for more information: See the user’s manual of Leica, model: RM2265).
    13. Take out the paraffin block of a tissue specimen from the mold manually, trim the paraffin block, and fix it in the specimen clamps and holder in the microtome (Supplemental Figure 1H). This step will assure a clean and homogenous cut with the formation of a straight ribbon, as shown in the Supplemental Figure 1K.
      Trim the paraffin block to get the cutting surface of the specimen facing the knife with a microtome (Supplemental Figure 1I), if you need it.
    14. Capture the sections by using a brush (Supplemental Figure 1K) and put them on a poly-L-lysine treated microscope slide (Supplemental Figure 1L) or 8-well ibidi µ-slides (Supplemental Figure 1M) without inverting sections, i.e., position tissue section side that was facing the knife (the "shiny" side) on the charged slide or ibidi well bottom glass.
    15. Add a droplet of water between the slide and the serial tissue sections on the slide or a single ibidi well (Supplemental Figures 1N and O, see Note below).
      Note: If you use the ibidi multi-well slides for dSTORM sample preparation, add 0.1% Poly-L-Lysine solution instead of water at this stage.
    16. Keep the slides at 42 °C for 3 min.
    17. Remove the water carefully without touching the cuts. Any drop of remaining water will form a bubble and will lead to the loss of this part of the sample. At this stage, manually spin-dry the slide by swinging it at arm's length 1 or 2 times (Supplemental Figure 1P).
      Note: Wearing a face mask and gloves is necessary only during the pandemy.
    18. Leave to dry at least overnight at 37 °C on a heating plate (Supplemental Figure 1Q). The slide can be conserved at room temperature for at least one month. Use the microscope slide boxes for the storage to avoid the dust.
    19. Deparaffining (Supplemental Figure 1R): Place up to 8 micro slides vertically (or 16 slides back to back) into a 250 ml Coplin glass staining jar and immerse the slide in the three successive treatments with 200 ml of Histoclear each for 30 min, at RT in normal light condition, shaking is not necessary. Then wash the Histoclear-treated microslides with a 200 ml of 100% ethanol using a 250 ml glass staining jar for 20 min.
      Note: The jar can be re-used at the next to the following step. Do not wash jar with water; just decant the solution from the jar.
    20. Rehydrate the samples with successive treatments using the same two glass jar used in previous steps, 15 min each, in 100% ethanol, 70% ethanol, 50% ethanol, 25% ethanol, 10% ethanol in 2F4 buffer, and finally 100% 2F4 buffer, at RT in normal light condition, shaking is not necessary. Use approximately 200 ml volume for each step.
      Note: The jar can be re-used at the next to the following step. Do not wash the jar with water; just decant the solution from the jar.
    21. Proceed to Step C1 (Immunolabeling) as soon as possible to avoid the drying of sections.
      Notes:
      1. In the Step B12, the sample should be prepared in advance and kept at 4 °C overnight. However, samples can also be cut (Steps B12-B18) 1 h after Step B10. In such case, store the samples at -20 °C for 1 h prior to cutting and cut in less than 15 min after taking the sample from -20 °C.
      2. Use Eppendorf tubes to store tissues after fixation at 70% ethanol and dehydration in 100% ethanol.

  3. Sample storage breakpoints: After the replacement to 100% Histoclear completely, the sample can be stored for a few days at RT. However, Histoclear is volatile and dissolves the paraffin; thus, it is not recommended for more than a few weeks. After fixation sample could be stored for months a 4 °C. After the embedding sample could be stored for years at a temperature below 25 °C. After cutting with microtome, the sample on microslides could be stored for several weeks at RT.

  4. Immunolabeling
    We present an example of multicolor immunostaining with 2F4 antibody against low methylesterified homogalacturonan binding a dimeric association of homogalacturonans through calcium ions, LM20 for high methylesterified homogalacturonan, JIM7 for partially methylesterified homogalacturonan, CBM3 and CBM4 to recognize mostly crystalline and amorphous cellulose respectively, PDM recognizing mannans, and LM24 recognizing xyloglucans. For all the steps, use 2F4 buffer instead of the PBS, even when not using the 2F4 antibody. It has proven to work correctly with different LM and JIM antibodies, for both CMB3 and CMB4 and microtubule antibodies (for the list of available LM, JIM and others antibodies and plant probes, please look here: www.plantprobes.net) using tissue cut samples of Arabidopsis, rice, star fruit, and maize (Yang et al., 2016). Importantly, if at any stage you wash out this buffer with water or PBS you will lose the 2F4 antibody staining.
    Notes:
    1. Prior to immunostaining, optionally, quench the free aldehyde groups using 50 mM NH4Cl in 2F4 buffer for 15 min. Wash 3 times with 2F4 buffer, each time 3-5 min. This step is recommended since any residual aldehyde group from FAA solution used at Sample Fixation Step B2 will react with the amino group of the antibodies leading to unspecific antibody binding.
    2. We recommend applying all the primary antibodies successively as described in Steps C1-C5, to prevent competition and steric hindrance effects, especially for the closely located epitopes, such as cell wall targets presented here (cellulose, xyloglucan, pectin and mannan, Figure 1). However, performing simultaneous primary antibody incubation works well in most cases. 
    3. To avoid the unspecific antibody binding, we use 5% milk in the 2F4 buffer as a blocking buffer. However, the milk can be contaminated over time; therefore, complete the immunostaining protocol Steps C1-C5 in less than 72 h, and where necessary, perform the overnight antibody incubation at 4 °C. Perform short antibody incubation and washing steps at room temperature (RT).
    4. Time schedule for staining. For the thin tissue sections (< 5 µm) 2 h at RT primary antibody incubation time is sufficient. For the thicker cuts and for certain antibodies, this time is extended to a minimum 3 h.
    5. Microwave Treatment. Depending on the manufacturer, selected antibodies may need microwave heating for activation or reaction acceleration (1 to 2 min at 400 watts). This step should be fast to prevent sample boiling–none of the antibodies presented in this protocol display improved efficiency of labeling after microwave heating.
    6. For enzyme treatments in the case of Aspergillus pectolyase from Sigma, treat sections with 200 μl of pectolyase in the incubation buffer (see Recipes) at the final dilution 0.1% at room temperature for 10 min prior to Step C1 (CBM incubation) or Step C2 (primary antibody incubation). After the enzyme treatments, wash sections with 2F4 buffer 3 times each time 3-5 min. When you use another pectate lyase such as Megazyme E-PLYCJ, check their publica- tion list for the reaction condition.
    7. How to make the hand made humid chamber (Supplemental Figure 1U). Place 2-3 layers of paper towel on the bottom of an empty micropipette box (10 cm x 14 cm x 9.5 cm) or an empty box for the microscopy slide tray (3 cm x 8.5 cm x 21 cm) and add distilled water (~20 ml), so that paper towels are humid. Cover the box. Use aluminum foil to wrap the humid chamber. If you use micro slides for confocal imaging sample preparation, make sure that the sample on the microslide is not in contact with the wet paper towels. For this, we recommend a microscopy slide tray.
    At each step, we use 200 µl of the antibody-containing solution per coverslip (Supplemental Figure 1S), while we use 50 or 70-100 µl of the antibody-containing solution per a single well of the 8-well ibidi slide (Supplemental Figure 1T, see also Step C8). For washing, we use approximately 500 µl per coverslip or 200 µl per single well of 2F4 buffer with 5% milk. Position the sections in the center of a droplet. On the droplet, the reagents (antibodies) are concentrated (a process known as coffee stain). Here we present the following optimal sequential antibody staining order. Optionally, prior to the antibody or reagent incubation, Steps C1 or C2, incubate your sample in a blocking buffer solution for 30 min. Then go directly to Step C1.

    1. The cellulose-binding molecule (CBM3 or CBM4) incubation: Dilute 2/100 volume per volume (v/v) CBM3/4 reagent in blocking buffer and add to samples. Incubate for 2 h at RT or overnight at 4 °C in the humid chamber(Supplemental Figure 1U). After removal of CBM3 solution, wash 3 times for 5 min using the blocking buffer. If you perform overnight incubation at 4 °C, the next day, take the humid chamber for samples out of the fridge at least 30 min before the washing steps.
    2. The first primary antibody incubation: Dilute 20/100 v/v 2F4 antibody in blocking buffer and add to samples. Incubate for 2 h at RT or overnight at 4 °C in the humid chamber. After removal of 2F4 solution, wash 3 times for 5 min using the blocking buffer. If you perform overnight incubation at 4 °C, the next day, take the humid chamber for samples out of the fridge at least 30 min before the washing steps.
    3. The second primary antibody incubation: Dilute 10/100 v/v LM20 antibody in blocking buffer and add to samples. Incubate for 2 h at RT or overnight at 4 °C in the humid chamber. After removal of LM20 solution, wash 3 times for 5 min using the blocking buffer. If you perform overnight incubation at 4 °C, the next day, take the samples out of the fridge at least 30 min before the washing steps.
    4. The third primary antibody incubation: Dilute 1/100 v/v rabbit or chicken anti-his tag antibody in blocking buffer and add to samples. Incubate for 2 h at RT or overnight at 4 °C in the humid chamber. After removal of the antibody solution, wash 3 times for 5 min using the blocking buffer. If you perform overnight incubation at 4 °C, the next day, take the humid chamber for samples out of the fridge at least 30 min before the washing steps.
    5. The secondary antibody incubation: Dilute all of three secondary antibodies 1/100 v/v in the same blocking buffer as a secondary antibody-mixture. Incubate for 2 h at RT or overnight at 4 °C in the humid chamber. Incubate the secondary antibodies in the dark to avoid the photo-bleaching. For this, wrap the humidified chamber in aluminum foil. After removal of the secondary antibody-mixture solution, wash 3 times for 5 min using the blocking buffer. If you perform overnight incubation at 4 °C, the next day, take the humid chamber for samples out of the fridge at least 30 min before the washing steps. The secondary antibody incubation could also be done sequentially (see Troubleshooting subsection), although in the case of Figures 1 to 4, all the secondary antibodies were added simultaneously.
    Notes:
    1. Steps C1-C5: Perform all the antibody incubation steps in the humidified chamber.
    2. This protocol can be extended for more than three targets using the antibodies from another species, such as rabbit, guinea pig, horse, and human. However, imaging more than four colors on the confocal microscope requires additional laser lines (> 700 nm) or spectral unmixing procedure.
    3. Make negative control images without 1st, 2nd, and 3rd primary antibodies like Figure 1A (Only Step C5 with secondary antibody mixture, at least in the case of your first trial.
    4. For dSTORM imaging, from Step B14, we recommend using the multi-well Ibidi Glass Bottom µ-slides, such as 8-well slides (Ibidi), which can be easily inserted and clipped on many microscope stages. The multi-well slides are convenient for the preparation of several imaging conditions, reduce the quantity of reagent used, and improves the homogeneity of the immunostaining for the semi-quantitative analysis. For 8-well Ibidi slides, we recommend using 70-100 µl of reagent per well at Steps C1-C5 of immuno-labeling.

    1. Post-Immuno fixation.
      This step is important for the dSTORM nanoscopy samples and is not necessary for samples prepared for confocal imaging using mounting media. Since dSTORM samples, contrary to confocal microscopy samples, are not mounted in the mounting media, it reduces the thermal motion of the antibody complex and slows down the antibody dissociation from the epitope.
      1. Incubate the sample for 10 min in 3.7% formaldehyde (see Recipe 4) diluted in an appropriate amount of 10x 2F4 buffer and distilled water at RT. For this, place 200 µl drop of 3.7% formaldehyde solution on the sample located inside the ibidi multi-well slide. Perform this step under the fume hood and wear nitrile gloves.
      2. Wash 3 times for 3 min with the 300 µl of 2F4 buffer. Perform this step under the fume hood.
      3. Quench the formaldehyde with 70 µl drop of 50 mM ammonium chloride for 15 min. This step reduces the risk of contact with formaldehyde to your skin while handling the sample during the imaging. Dilute the ammonium chloride in 2F4 buffer (see Recipes). After incubation, wash briefly 3 times with the 300 µl drop of 2F4 buffer. You can also use other aldehyde quenching reagents containing amine groups, such as 50 mM of glycine in 2F4 buffer.
    2. Sample mounting and storage. Only for confocal imaging.
      Note: Do not perform this step for dSTORM sample preparation.
      To extend the lifespan of the sample, mount the slide with a coverslip in mounting media containing antifade reagent. We suggest ProLong Gold Antifade Mountant (Thermo Fisher Scientific) (Supplemental Figure 1V). To prevent the formation of bubbles, place a droplet of mounting media on the coverslip, and gently lower it on the slide using a micro tweezer (Supplemental Figure 1W-AA). Seal the coverslip with nail polish to prevent drying (Supplemental Figure 1AB) and put sealed micro slides into Cardboard Slide Tray (Supplemental Figure1AC) and store in the dark at 4°C.
    3. Sample storage. Only for dSTORM imaging.
      After completion of Steps C6, add 500 µl of 2F4 buffer to each of 8 wells, seal with parafilm, wrap in the aluminum foil, and keep in a humid chamber at 4 °C as presented in Supplemental Figures 1AD and 1AE.
      Note: You can perform this for confocal imaging, but then you cannot mount your sample in the antifade mountant; therefore, it is not recommended.
      Choosing the fluorescence dyes.
      For confocal imaging, we recommend using bright and photostable dyes, such as Alexa Fluor, ATTO, and CF series. For the dSTORM imaging, only certain dyes work. We recommend ATTO488, CF568, and Alexa647 dyes. For the dSTORM nanoscopy imaging, we recommend using F(ab’)2 secondary antibody fragments or nanobodies, or, when possible, to conjugate the primary antibodies directly with the fluorophore of interest. For densely packed epitopes, we recommend using either single labeled secondary antibodies.

    Troubleshooting
    High background: Reduce secondary antibody concentration. The good practice is to perform the titration of both primary and secondary antibodies. If, for instance, the recommended antibody dilution is 1:200, a good starting point for the titration test is 1:50, 1:100, 1:200, 1:400, and 1:800 dilution. Before immunostaining, quench free aldehyde groups using 50 mM NH4Cl in the 2F4 buffer for 15 min. Use serum in blocking buffer in which the secondary antibody has been raised, e.g., goat serum for antibodies produced in goat. Incubate the antibodies at 4 °C overnight prior to Steps C1 or C2.
      A high degree of colocalization when it is not expected: Use sequential staining for all the secondary antibodies. If possible, use the same host species for all the secondary antibodies (For example, a set of anti-rat antibody, anti-rabbit antibody and anti-mouse antibody from goat and so on). To avoid off-target binding, use highly cross-adsorbed secondary antibodies. We recommend preparing a test sample for which only Step C5 is performed, which is the application of the secondary antibodies without primary antibodies.
      Low signal: Increase the primary antibody concentration and/or incubation time. Try to use a fresh batch of the primary and secondary antibodies. Change the secondary antibody (conjugated-dye, host species, or producer for the same type of antibody). Increase the secondary antibody concentration (perform the titration-test at the 2, 4, and 6 fold increased antibody dilution) and/or incubation time (perform tests at 2 h, 4 h, 6 h). Check the pH of the 2F4 buffer. If pH is not 8.0, adjust it.

    Confocal imaging tips
    1) Counterstaining
    For the semi-quantitative analysis of the cell wall component with a confocal microscope, compare the samples from the same experimental preparations. If possible, consider the observation of counterstained sample with the third or fourth channel of the laser scanning confocal microscope, if the counterstaining intensity is expected not to change in different sample conditions. For the cell wall staining, use, for instance, calcofluor white stain. Add 200 µl of calcofluor white stain after the Step C5 at 5 mM concentration in 2F4 buffer and incubate for 5 min at room temperature. Wash briefly twice with 500 µl of 2F4 buffer before applying the antifade mounting media to the sample (Step C7). The counterstaining is useful to normalize the intensity data to size, e.g., area or the cell wall thickness. Prepare all the conditions you want to compare for each experimental preparation.
    2) Operation of microscope
    Switch on the laser 1h before imaging. During the imaging, make sure not to saturate the signal at the detectors for downstream quantification. Keep the laser power, and detector gains the same across the experiments and keep all other settings constant between comparable experiments. Additionally, in order to avoid the photobleacing during the optimization of the image quality before the final take, consider using a tissue sample or region with less scientific interest, but a similar signal level present on your slide, 1) Find the target point and focus the section in bright field. Differential interference contrast image (DIC) is more convenient to focus the image if DIC is installed to your microscope. 2) Optimize the image quality at the position near the final target point. 3) Stop scanning frequently during the optimization of the image quality. 4) As the initial settings for confocal laser scanning mode, 1 to 2% of laser power is a good starting point to focus on a sample. 5) Faster or maximal scan speed (Arbitrary level of scan speed: > 9 in the case of Zeiss LSM 710 ) is recommended to focus on the initial conditioning (Live button of ZEN software is convenient in Zeiss LSM 710 ).
    Note: There are fifteen and arbitrary levels of scan speed in LSM710 and the absolute speed at maximum level “15” is 8 frame/s, according to Zeiss local office. When performing quantitative imaging, due to photobleaching, never take the final image of the same area twice. Therefore, move the X-Y-Z stage for the micro slide in a certain direction (from right to left and from front to back) to avoid the observation of the same point. For reliable quantification, 2-3 independent experiments (independent technical replicates) need to be carried out and compared. Perform a pilot experiment to determine the common parameters, such as gain and pixel dwell time, and to abolish/minimize any photobleaching. Sequentially image all the channels, always starting with the longest wavelength and ending with the shortest wavelength.

Data analysis

Figure 1 shows dual- and triple-color IHC of different cell wall epitopes, representing members of three different families of the cell wall polysaccharides: pectin (2F4, LM20 , and JIM7 ), cellulose (CBM4, CBM3) and hemicellulose (PDM and LM24 ) in the transverse section of star fruit leaf rachis (Supplemental Figure 2, and Table 1).
  Comparing Figure 1 panels A and C, the cellulose staining (CBM3 and CBM4, see Table 1) is weak in areas presenting high HG counts and high in tissues with low HG counts. This could suggest that cellulose and pectins occupy exclusive wall compartments. However, the enzymatic extraction of pectin revealed much higher cellulose detection levels with CBM4 (compare Figures 1C and 1E). This indicates that, in the intact cell walls, the majority of cellulose epitopes are masked by pectins. Without pectin extraction, the remaining cellulose staining may correspond to xyloglucans, which are partially detected by CBM probes (see PlantProbes CMB3 reagent description, Hernandez-Gomez et al., 2015). Indeed, Figure 1D shows that CBM3 and LM24 antibody against xyloglucans present a low degree of overlap in the subset of walls.


Figure 1. Multicolor confocal imaging of different cell wall epitopes in the star fruit (Averrhoa carambola) leaf rachis. A. Control images with only the secondary antibody staining. B. Triple staining of 2F4, CBM4 and LM20 , and (C) 2F4, CBM4, and JIM7 epitopes. D. Double staining of CBM3 and LM24 . E. Double staining of CBM4 and PDM after enzymatic pectin extraction. All the images were acquired with the same microscope settings (see Table 2 for suggested settings). The last column represents transmission images of the cuts. Images were visualized in Fiji. Scale bars, 100 µm.

Table 1. The primary antibody, CBM reagents, and the secondary antibody dilutions used in Figures 1-4


Table 2. The suggested emission filter bandwidth for the triple immunostaining and a counterstaining with calcofluor white using confocal laser scanning microscope optimized to minimize spectral crosstalk. For details on the filter sets for dSTORM please refer to Haas et al. (2020)


Figure 2 shows the 3D dSTORM nanoscopy imaging of the different pectin species in the L2 layer (layer below the epidermis) of the Arabidopsis cotyledon with the longitudinal section described in Supplemental Figure 2E. In Figures 2A and 2B, the typical cell edge staining of low-methylated (2F4), high-methylated ( LM20 ), and partially methylated ( JIM7 ) pectin is observed (see Table 1). The 3D dSTORM images revealed that the staining is weak inside the cell wall (middle lamella) and is mostly limited to the cell wall edges facing the cytoplasm–this highlights one of the biggest limitations of IHC in general: the epitope accessibility and antibody penetrability. Yet, in light of available techniques that possess similar spatial and chemical resolution (e.g., iEM), 3D dSTORM provides overall higher labeling and detection density, and the number of epitopes detected in the middle lamella remains higher for 3D dSTORM than iEM (Cosgrove and Anderson, 2020). One possible way to bypass this limitation is to remove one polymer (for example, structural protein degraded with the recombinant Protease K) from the cell wall to increase antibody penetration. However, this may cause an unspecific wall perturbation. The development of small nanobodies against the cell wall targets will help in the future to address these limitations. The advance in correlative super-resolution light and electron microscopy may help enhance the performance and annihilate the limitations of 3D dSTORM and EM.


Figure 2. Two-color 3D dSTORM imaging of Homogalacturonan at tri-cellular junctions of L2 (subepidermal cell) layer in Arabidopsis cotyledon. Two-color scatter plots showing the 3D coordinates of localized (A) JIM7 (green) and 2F4 (violet), and (B) LM20 (green) and 2F4 (violet) epitopes. Image insets show a low-resolution oblique illumination image with red square outlining regions shown in the scatter plots. 2D–two dimensional, top view (XY), and 3D–three dimensional, inclined view, where the Z-axis is oriented perpendicular to the conventional image plane. The orange lines show the cell wall region enlarged in the panel below. Image scale bar, 4 µm. Data were visualized using GrafeoV.2.

Figure 3 shows the 3D dSTORM nanoscopy detection of the different pectin species in the epidermal layer of the Arabidopsis cotyledon with a longitudinal section described in Supplemental Figure 2G. In both Figures 3A and 3B, 2F4 and LM20 epitopes form a filamentous pattern of staining in the anticlinal, but not periclinal walls, described previously as the HG nanofilament (Haas et al., 2020). This nanofilament quaternary structure is challenging the canonical view on the pectin in muro architecture as an amorphous gel-like matrix. These unexpected results raise two concerns: (1) that the filamentous pattern represents pectins as a spacer between cellulose microfibrils, or (2) that pectin antibody cannot penetrate between the grooves formed by the cellulose microfibrils (Cosgrove and Anderson, 2020). To address these concerns, we labeled partially methylated HG epitope ( JIM7 ) in the anticlinal walls of the cotyledons, Figure 3A. JIM7 presents a much broader and uniform distribution pattern compared to LM20 and 2F4 HG epitopes. First, the diffuse distribution of JIM7 epitope compared with LM20 /2F4 filamentous distribution suggests that the pectin methylation pattern somehow correlates with its localization. Secondly, it implies that HG nanofilaments of LM20 and 2F4 are likely, not due to epitope inaccessibility in the grooves formed by cellulose microfibrils, as JIM7 epitope can be localized there, Figure 3A. Moreover, double staining for cellulose (CBM3) and HG (2F4) in Maize epidermal cells of an immature leaf at 6th node (longitudinal section in Supplemental Figures 2C and F), without prior extraction of pectins, shows that empty spaces between 2F4 nanofilaments are not filled with cellulose and that the overall level of the cellulose staining without pectin extraction is very low, Figure 3C.


Figure 3. Two-color 3D dSTORM imaging of Homogalacturonan at the lobes of Arabidopsis cotyledons pavement cells and maize leaf pavement cells. Two-color scatter plots showing the 3D coordinates of localized (A) JIM7 (green) and 2F4 (violet), and (B) LM20 (green) and 2F4 (violet) epitopes in the Arabidopsis cotyledons. Image insets show a low-resolution fluorescence image of the regions shown in the 3D dSTORM scatter plots. The orange lines show the cell wall region enlarged in the panels below. (C) Two-color scatter plots showing the 3D coordinates of localized 2F4 (violet), and CBM3 (orange) epitopes in the Maize leaf. Scale bar, 1 µm. Data were visualized using GrafeoV.2.

Figure 4 shows two-color 3D dSTORM imaging of Arabidopsis primordia and meristem directed towards cellulose (CBM3 or CBM4) and (hetero)mannan (PDM) and with prior pectin extraction with a longitudinal section of 1 cm of the inflorescence (Supplemental Figures 2B and 2D, Table 1). Comparing panels A and B show that CBM3, detecting predominantly crystalline cellulose, forms discreet filaments distribution as contrasted with CBM4, tagging amorphous cellulose. The staining for mannans displays different abundance in different cell walls: from extended wall sections mostly exclusive from the cellulose (Figure 4A) to punctate staining decorating extremities of the cellulose nanofibrils (Figure 4B).


Figure 4. Two-color dSTORM imaging of cellulose (CBM3/4) and (hetero), mannan (PDM) in the Arabidopsis primordia, and meristem. Pectins were enzymatically extracted with pectolyase treatment prior to the immunostaining protocol (Step C1). Two-color scatter plots showing the 3D coordinates of localized (A) CBM4 (blue) and PDM (orange) in a meristem, and (B) CBM3 (blue) and PDM (orange) epitopes in primordia. Insets on the left of the plots show a low-resolution fluorescence image with a red square outlining regions presented in the scatter plots. Scale bar, 1 µm. Data were visualized using GrafeoV.2.

Conclusion
IHC is a powerful tool to study subcellular changes in cell wall chemistry. However, it has several limitations. The number of epitopes that could be explored simultaneously is limited (< 4) due to spectral cross-talk and the number of the antibody species. In addition, only epitopes with well-defined antibodies can be detected. Furthermore, the accessibility of the antibody to the inner cell wall components is limited, except if one of the cell wall components is degraded to help penetrability. Also, the sample preparation for IHC may cause tissue distortion; therefore, spatial scale measurements and topology may not be absolute. Despite this, multicolor IHC coupled with a novel optical nanoscopy offers unprecedented insights into the complex spatial organization of biomolecules. In combination with super-resolution imaging, multicolor IHC is now changing our vision on the cell wall architecture and plant growth. The cell wall mediates plant cell interactions with the environment, and therefore we expect this technique will help us understand plant immunity, fruit ripening, plant-microbe interaction, plant growth, and yield. Constant improvements in fluorescence probes, single-molecule techniques, and data analysis make that 3D dSTORM has huge potential to refine our knowledge on molecular assemblies and their function (Klein et al., 2014; Kim et al., 2019; Gwosch et al., 2020; Zhang et al., 2020). Hence, “Seeing, contrary to popular wisdom, isn't believing. It's where belief stops, because it isn't needed any more”, Terry Prattchet, Pyramids.

Recipes

  1. 2F4 Buffer (T/Ca/S buffer final concentration)
    To dilute all the antibodies, prepare blocking buffer and washing buffer (http://www.plantprobes.net/pp_2F4.pdf).
    Tris-HCl 20 mM pH 8.2
    CaCl2 0.5 mM
    NaCl 150 mM
    The final pH should be 8.0
  2. Formaldehyde Alcohol Acetic Acid FAA solution
    50% ethanol
    10% acetic acid
    3.7% formaldehyde
    The percentage indicates the final concentration in FAA solution
  3. 1 M ammonium chloride solution diluted in 2F4 buffer
    Mix 53.489 g of ammonium chloride powder in 1 L of 2F4 buffer
    Adjust the pH to 8.0
  4. Formaldehyde diluted in10x 2F4 buffer
    Mix 1 volume of 37% formaldehyde solution, 1 volume of 10x 2F4 solution, and 8 volumes of distilled water.
    Perform under the fume hood
  5. Citrate-Phosphate Buffer for Pectolyase incubation (pH 4.8, Stoll and Blanchard, 1990)
    1. 0.2 M Na2HPO4·7H2O
      Dissolve 53.65 g in MilliQ water and make 1 L of solution A
    2. 0.1 M citric acid
      Dissolve 19.21 g in MilliQ water and make 1 L of solution
    3. Mix 24.8 ml of solutionA and 25.2 ml of solution B and dilute to a total of 100 ml for pH 4.8
      If you need, check pH value

Acknowledgments

This work presents an extended protocol, which was published in Haas et al., 2020. The dSTORM was performed at the MRC Laboratory of Molecular Biology, Cambridge, and we thank Nick Barry and Jonathan Howe for their support. We Herman Höfte for help with the fundraising and discussion of the results.
  Funding: AP has received the support of the French National Research Agency (ANR) GoodVibration ANR-17-CE13-0007 and of the EU in the framework of the Marie-Curie FP7 COFUND People Program, through the award of an AgreenSkills+ fellowship (under grant agreement n° 201310). The IJPB benefits from the support of Saclay Plant Sciences-SPS (ANR-17-EUR-0007). The Microscopy Facility at the Sainsbury Laboratory is supported by the Gatsby Charitable Foundation. This work has benefited from the support of IJPB's Plant Observatory technological platforms.

Competing interests

The authors declare no competing interests.

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简介

[摘要]所述的植物细胞壁(PCW)是pecto该信封-cellulosic细胞外基质Ë S中的植物细胞。通过整合外和内-细胞线索,PCW介导的基本的生理功能太多了。值得注意的是,它可以通过调节其局部机械化学特性来控制组织的生长。为了完善我们的PCW的这些基本性能的了解,我们需要为本地(准确观察一个合适的工具在穆罗的细胞壁组分)的结构。使用无标记技术,例如AFM,EM,FTIR和nd拉曼显微镜。但是,它们没有化学或空间分辨率。免疫标记随e lectron米icroscopy允许细胞壁纳米结构的观测,但是,它主要局限于单一和,较不频繁地,多重标记。我免疫组织化学(IHC)是分析低压配电设计的多功能工具在组织重刑和多种生物分子的定位。可以用标准的衍射极限光学显微镜观察细胞壁成分化学变化的亚细胞分辨率。此外,新颖的化学成像工具(例如多色3D dSTORM (三维直接随机光学重建显微镜)纳米显微镜)可以以纳米精度和三维精度解析细胞壁聚合物的天然结构。

这里,我们提出用于制备以与实施例的PCW组分的多目标免疫染色的协议拟南芥吨haliana ,杨桃(阳桃),和玉米薄的组织切片。该协议与标准共聚焦显微镜dSTORM纳米镜兼容,也可以用于其他光学纳米镜,例如STED(受激发射损耗显微镜)。该方案可适用于任何其他亚细胞区室,质膜,细胞质和细胞内细胞器。


[背景] PCW是一个复杂的材料,其是硬脑膜BLE但也经历响应于内部和外部刺激如组织膨胀或病原体攻击恒定结构变化。然而,这些看似矛盾的特征,机械强度和结构适应性如何协同作用,仍然是植物细胞生物学中尚未解决的问题。PCW包含纤维素,半纤维素,果胶的不同变体和各种蛋白质,其结构高度组织化(Peaucelle,2018)。果胶家族由几种聚合物组成。Ť他最为丰富,homogalacturonan,可细胞壁插入后脱甲基化。这种化学变化是细胞伸长,分化和定向生长过程中的重要一步(Peaucelle等,2012)。有几条证据表明,形态发生和细胞分化取决于细胞壁化学和细胞壁聚合物组织的局部变化(Yang等人,2016; Anane等人,2017; Zhao等人,2019; Haas等人。(2020年)。因此,对PCW组件架构的详细了解对于理解植物生长至关重要。从历史上看,已经使用生化方法研究了细胞壁的结构,包括分解组织并破坏其聚合物的天然组织(Höfteand Voxeur,2017)。其他成像模态,诸如电子米icroscopy (EM) (安恩等人,2017)和原子力显微镜(AFM) (张等人,2017)被用于原位细胞壁观察,但是这些技术往往缺乏化学相反,仅提供相关的量化。多色免疫组织化学(IHC)可以揭示组织切片内的多个靶标,并将它们的空间组织分解为三个维度。尽管它在细胞生物学领域得到了广泛的应用,但多色IHC仍不是研究细胞壁的标准工具。针对细胞壁靶标的抗体和探针种类繁多,再加上多色IHC,以及dSTORM (< 40 nm)的高分辨能力,可以对细胞壁纳米结构进行定量原位化学分析。还存在用于组织切割的细胞壁分析的其他成像技术,例如Raman (Wightman等人,2019)和FTIR (Mravec等人,2017; Cuello等人,2020)。这些技术基于不同化学成分的特征吸收/传输,可以在细胞水平上测量细胞壁组成的某些变化,但可能缺乏敏感性,并且它们在亚细胞水平上观察变化的能力受到严重限制。

所述dSTORM允许与在5-10nm精度生物分子的定位; 但是,最终dSTORM分辨率通常在40 nm左右,受限于抗体复合物的大小(约15-30 nm)。免疫金电子显微镜(iEM )也可与ICH结合使用,以高分辨率研究细胞结构。IEM是堪比dSTORM分辨率,并且还通过抗体复合物的限制大小。纳米金颗粒的大小决定了iEM的对比度和分辨率;> 1 nm的金纳米颗粒可用;然而,如此小的颗粒限制了对比度,并且经常使用较大的颗粒。与dSTORM相比,IEM具有几个其他缺点:(1)用蛋白A(或G)金络合物探测的一抗不穿透树脂包埋的样品,仅识别表面表位,尽管是连续和超薄的低温切片技术可以解决这个问题(50 nm,-120 °C [ Slot,1989 ] );(2)样品大多是单标签的;然而,通过使用不同的纳米颗粒大小,可以标记两个或三个表位,但这需要超薄冷冻切片技术的技术经验(Slot和Geuze,2007)。(3)IEM的标记和检测效率低(比dSTORM低3-5个数量级(Majda等,2017; Haas等,2020)),这也与以下事实有关,即仅表面抗原决定簇是因此,多色3D dST ORM纳米技术可以提供超越以前的技术应用范围的对细胞壁多糖天然结构纳米结构的空前见识,DSTORM可以对细胞进行三维(3D)定量纳米成像和组织(Heilemann等,2008;黄等,2008;范·德林德等。,2011; Sydor 。等人,2015年,徐等人,2018) 。主要适用于蜂窝系统,它推出了新的蛋白质的结构组织,例如突触纳米域,反突触纳米柱,DNA重组酶纳米丝,核被膜孔结构,细胞骨架,线粒体,粘附复合物和染色质转录景观(Shroff等,2008; Shim等,2012) ;Löschberger等。,2012;Jakobs和Wurm,2014年;Prakash等。,2015;Boettiger等。,2016;Sellés等。,2017; Dlasková等。,2018; 哈斯等。,2018 a和2018 b ; Pan等。,2018; 夏等。,2019; Chen等。,2020; Wäldchen等。(2020年)。然而,由于需要组织水平成像的单细胞模型系统的局限性,几乎没有在植物科学中使用它(Liesche等人,2013; Komis等人,2015; Haas等人,2020)。在这里,我们介绍了与植物薄组织切片上的标准共聚焦显微镜和dSTORM纳米镜兼容的样品制备的详细协议。

关键字:植物细胞壁, 免疫组织化学, 细胞壁多糖, 超分辨显微镜, 3D dSTORM纳米显微技术, 形态发生


材料和试剂
 
1.用于dSTORM样品制备和成像的8孔ibidi µ-载玻片(Ibidi ,目录号:80827)      
2. 42 x 28 x 6 mm的活检盒(Leica Biosystems,ID:IP-活检盒-III)      
3.金属基础模具(Leica Biosystems,ID:金属基础模具)      
4.通过聚-L-赖氨酸处理的带电载玻片,用于共聚焦成像样品制备和成像,例如ProbeOn Plus(Fisher Scientific目录号:22-230-900或Polysine TM显微镜粘附载玻片(Thermo Scientific目录号:J2800AMNZ))      
5.铝箔(您可以从当地商店购买)      
6.该玻片,矩形,24×60毫米,#1.5厚度(Knittel玻璃,目录号:425-2460)      
7.容量为1.5 ml或2 ml的Eppendorf Safe-lock微量离心管(Eppendorf,目录号:022-36-320-4 [1.5 ml],022-36-335-2 [2 ml])      
8.纸板滑动托盘(Heathrow Scientific ,目录号:HD9902)      
9.纸巾(Divers / Dutcher,目录号:475040)      
10.空的移液器吸头盒,用于放置ibidi µ- slide的自制加湿室,例如Adamas-Beta 1,000 µl容量吸头   
11.丁腈手套,例如Fisherbrand TM Comfort丁腈手套(Fisher Scientific,目录号15642367 )   
12.国家诊断TM Histoclear TM组织清除剂(Fisher Scientific公司,HS-200-1GAL ,CAS号:5989-27-5)   
13.包埋中石蜡(Leica Biosystems,ID:em-400-embeddding-medium-paraffin )   
14. Oxoid脱脂奶粉(Thermo Fisher Scientific,目录号:LP0031B)   
15. CMB3a结晶纤维素结合模块,带有组氨酸标签的重组CBM蛋白(PlantProbes ,目录号:CMB3a)(Blake等,2006; Hernandez-Gomez等,2015),储存在-20 °C   
16.抗低酯化高半乳糖醛酸聚糖(甲基酯化度(DM)高达40%)的小鼠单克隆抗体2F4(PlantProbes ,目录号:2F4)(Liners,Thibault和Van Cutsem,1992),储存在4 °C   
17.针对高酯化同型半乳糖醛酸聚糖LM20的大鼠IgM单克隆抗体(PlantProbes ,目录号:LM20)(Verhertbruggen等,2009),储存在4 °C   
18.针对高半乳糖醛酸聚糖的部分甲基酯化表位的大鼠IgA单克隆抗体(PlantProbes ,目录号:JIM7 )(Knox等,1990),储存在4 °C   
19.针对木葡聚糖的鼠IgG2a单克隆抗体优先结合木葡聚糖LM24的XLLG基序(PlantProbes ,目录号:LM24)(Tanackovic等,2016),储存在4 ℃下   
20.鸡肉中生产的抗His标签多克隆抗体,抗6X-His标签(Abcam,目录号:ab9107)在-20 °C下储存   
21.在兔中生产的抗His标签多克隆抗体(Merck,目录号:SAB4301134 ),储存在-20 °C   
22. Paul Dupree赠送了一种抗曼南的兔PDM抗体(Handford等,2003; Yang等,2016),在4 °C下保存。有关可用性,请联系raymond.wightman@slcu.cam.ac.uk   
23.重组和His-标记的CBM4纤维单胞FIM我(ATCC 484)内切葡聚糖酶C(CBD4 N1产生自)E. Ç OLI。CBM4是哈里·吉尔伯特(Harry Gilbert)赠予的一种礼物(Johnson等,1996; Blake等,2006),储存在4 °C下。有关可用性,请联系raymond.wightman@slcu.cam.ac.uk   
24.山羊抗大鼠CF568二抗(西格玛,目录号:SAB4600086),储存在-20 °C   
25.山羊抗小鼠CF568共轭的F(ab)'2二抗片段(Sigma,目录号:SAB4600400),储存在-20 °C   
26.与Alexa Fluor 647(Stratech Scientific,目录号:115-607-003-JIR)偶联的山羊抗小鼠F(ab')2二抗片段,储存在4 °C下   
27.与Alexa Fluor 647(Abcam,目录号:ab181292)偶联的驴抗小鼠F(ab')2二抗片段,储存在4 °C下   
28.与Alexa Fluor 647偶联的驴抗鸡F(ab')2二抗片段(杰克逊免疫研究,目录号:703-606-155),储存在4 °C下   
29.与ATTO488偶联的山羊抗小鼠F(ab')2二抗片段(Hypermole ,目录号:2402-0.5MG),储存在-20 °C   
30.山羊抗小鼠Alexa 488共轭二抗(Sigma,目录号:SAB4600387),储存在4 °C下   
31.与Alexa Fluor 647(Abcam,目录号:ab150151)偶联的驴抗大鼠F(ab')2二抗片段,储存在4 °C下   
32.的ProLong Gold抗淬灭封固(热Ficher科学,目录号:P36934),保存于4 ℃下   
33.氯化铵(NH 4 Cl)(Sigma-Aldrich,目录号:254134),在室温下存放   
34.在H 2 O (Merck,目录号:P8920)中的0.1%(w / v)聚赖氨酸溶液,储存在室温下   
35.无水乙醇(CARLO ERBA试剂,目录号:4146802),在室温下存放   
36.醋酸≥96%(AnalaR NORMAPUR ,目录号:20099.324),在室温下存放   
37.甲醛(西格玛奥德里奇目录号:F1635 ),在室温下存放   
38.拟南芥花序分生组织固定在1厘米长的阶段,和3日龄幼苗的子叶,叶轴节星的F瑞特(补充˚F igure 1AG ),未成熟的玉米叶在叶片6个节点(补充˚F igure 1AF )   
39. Ñ AIL抛光(你可以从当地的药店得到它)   
40.用于果胶酶促提取的果胶水解酶(西格玛,目录号:P3026或Magazyme,目录号E-PLYCJ)   
41.用于一般细胞壁染色的Calcofluor White Stain (Sigma Aldrich,目录号18909-100ML -F )   
42. 2 F4缓冲液(T / Ca / S缓冲液终浓度)(请参见配方),在室温下保存   
43. FAA解决方案(请参阅食谱),存储在RT   
44.甲醛在10x 2F4缓冲液中稀释(请参阅食谱)   
45.用于果胶裂解酶孵育的柠檬酸-磷酸盐缓冲液(pH 4.8)(请参见食谱)   
 
设备
 
一次性切片机刀(Microm Microtech ,目录号:F / MM35p)
微型镊子(Ideal- tek ,目录号5-SA)
250毫升玻璃矩形Coplin染色罐,带盖(Wheaton,目录号:900620)
Fisherbrand TM显微镜载玻片盒,用于自制加湿室,用于Polysine显微镜载玻片(Fisher Scientific,目录号:22363400 )
用于捕获由切片机获得的系列切割的刷子(补充图1K,来自当地文具商店的ny精细刷子,例如Etude,P10531.00,#2 )
HistoCore Arcadia加热石蜡包埋站(Leica,ID:14039357258)
徕卡EG F电动可加热钳
切片机(Leica,型号:RM2265)
有关更多信息,请参见用户手册。
冰箱4°C
孵化室,例如Selecta (设定为60 °C),不需要气流
实验室冷冻室(-20°C),例如Kirsch FROSTER LABO 330 ULTIMATE
电子实验室加热板(BIO-OPTICA米兰的SpA ,CA talog Ñ棕土:40-300-300)
实验室化学通风柜
 
对于成像步骤:
任何配备四根激光线的荧光显微镜都可以在UV(405 nm),绿色(488 nm),红色(561 nm)和远红色(633 nm)中进行检测(此处我们使用Zeiss LSM 710,Zeiss Oberkoch en德国)
手册可在此处获得。
规范
支架:倒置(带有侧面端口的Axio Observer Z1)
Z驱动和XY工作台(选件):电动XY扫描工作台,具有标记和查找功能(xyz )和平铺扫描(马赛克扫描);最小增量1μm
物镜:x10,x25,x40,x63,x100
激光:氩激光(458、488、514 nm),氦氖激光(633 nm),二极管激光405 nm和DPSS激光561 nm
405 nm for Calcofluor白色
Alexa 488和ATTO 488的488 nm
CF568和Alexa 568的561 nm
Alexa 647的633 nm
扫描模块
型号:具有32个光谱检测通道(QUASAR)的扫描模块
扫描仪:两个独立的振镜扫描镜,具有超短的线条和框架,可以向后飞。
扫描分辨率:4 X 1至6 ,144 X 6 ,144个像素
扫描速度:8帧/秒,512 x 512像素。
荧光光谱探测器数量:2
明场透射探测器:已安装
软件
标准软件:ZEN2010
可选软件战:Image J或斐济
计算机规格:HP Z800工作站,64位Windows 7旗舰版2009年,24 GB内存,英特尔®至强® CPU,X5650,两个处理器2.67 GHz的,2.66GHz的
野外显微镜(Nikon N-STORM)
 
软件
 
Grafeo(用于dSTORM数据分析和可视化的定制软件,https://github.com/inatamara/Grafeo-dSTORM-analysis-(Haas等人,2018 b )
斐济(https://imagej.net/Fiji/Downloads)
 
程序
 
在此协议中,我们使用拟南芥子叶分生组织,杨桃(Averrhoa carambola)rachis(复合叶中的中央纤维)和玉米叶样品。
样品制备
杨桃生长在温室中的土壤两年,和叶穗轴在收获四月2019玉米的乞讨土壤中生长在田间播种在五月和在七2019年开始收获其开花前。拟南芥物生长于MS(的Murashige和Skoog的)不含蔗糖固体营养琼脂培养基上,在21中恒定光℃下,和发芽(3 DAG)后收获在3天苗(Peaucelle,怀特曼和Höfte,2015) 。拟南芥分生组织是从生长在生长室内土壤上的植物中收获的,当花序长为1 cm时收获。去除具有可见萼片的花,保留所有最近的花蕾(如Yang等人,2016所述)。              
叶杨桃的羽轴切割用超薄切片机可有可无刀成0.5厘米长的外植体(补充连接古尔小号1AG ,2A )。玉米芽尖端的未成熟区域是从小植株中分离出来的(图1AF)。将在第6个节点没有中脉的玉米未成熟叶片切成5 mm x 5 mm的正方形(补充图2C )。
固定拟南芥不同器官的体积范围通常在1:10至1:100(组织至固定液体积)之间,此处我们将20颗未除去子叶的幼苗放在装有1.0 ml FAA溶液的1.5 ml Eppendorf管中。玉米叶外植体的体积比为1:3,杨桃叶ra轴外植体的体积比为1:100。我们分别在装有1.5 ml FAA溶液的2.0 ml Eppendorf管中放入5个玉米叶外植体和10个杨桃叶轴心外植体。的显著协议步骤示于补充图1和制备组织外植体,并在切片机切割位置补充˚F igure 2。
 
固定和样品嵌入
戴丁腈手套。
修复植物器官在˚F在1.5 / 2 mL Eppendorf管AA溶液在室温下1个小时或过夜,在4℃。将固定的样品在4 ° C下的70%EtOH中保存1个月(s注释)。无需真空处理。
脱水的样品小号70%,95%,和两次100%乙醇:通过在连续稀释的乙醇,在室温下温育各至少30分钟。在Eppendorf管中使用约1.5 ml的体积。
在室温下,用1.5 ml 50%Histoclear的乙醇在Eppendorf中的乙醇中替换乙醇1小时,然后在室温下分别用1.5 ml 100%Histoclear的乙醇替换1 h(此时间取决于样品的厚度。减少孵育时间)适用于非常薄的组织切割,例如根部或下胚轴切割至30分钟)。
将样品转移到一个活检盒(IP-活检盒式-III,徕卡Biosystems)中。在步骤B前12小时,在预热至60-70 ° C的培养箱中开始熔化石蜡。完全熔化石蜡和新Histoclear应预热至60-70℃之前小号TEP乙6,使50%的石蜡在Histoclear 。在一个典型的实验中,我们在S tep B 6中使用大约600 ml的石蜡和100 ml的Histoclear 。
预热吨他含有200ml最终的250ml玻璃广口瓶中的50%Histoclear和50%石蜡之前小号TEP乙6。
注意:我们建议使用熔点低(56-57 ° C)的EM-400包埋中等石蜡。这有助于精确定位样品并减少组织变形。
通过将活检盒浸入250 ml Coplin玻璃染色罐中(补充图1B),在培养箱中用石蜡代替Histoclear 。在60-70 ° C的温度下,先将200%的50%Histoclear和50%的石蜡的混合物在3个小时内混合3小时,然后再将200 ml的100%的石蜡进行两次混合,持续3 h,最后在60- 70 ℃ 。
开启HistoCore Arcadia H加热石蜡包埋位置(补充图1C),并在步骤B 8之前将HistoCore Arcadia H设置为操作模式(将石蜡罐,分配器,工作表面的温度设置为〜70 ° C并设置一个冷点)在4 °C下。有关更多信息,请参见HistoCore Arcadia H用户手册)。
从培养箱中取出活检盒并在250ml Coplin玻璃染色罐中加热100 %石蜡。在室温下固化石蜡之前,让活检盒漂浮在加热的仪器托盘融化的石蜡中,然后将其放置在HistoCore Arcadia H约70 ° C的热准备表面上(补充图1D)。
使用可加热的镊子将样品从活检盒转移到金属基础模具(补充图1E)。样品应被定位在垂直于切割平面在室温下固化的石蜡之前(补充Figu水库1F至1H和2 d )。
一旦定位,通常可以在4°C下使用冷点(补充图1G中所示的小圆形金属板)实现石蜡的快速固化。
注意:石蜡的快速冷却将限制石蜡晶体的形成,并使制剂透明。相反,缓慢冷却将导致晶体形成和制剂的扩大。结晶的石蜡稍硬一些,极大地限制了切片机切割过程中定位样品的能力。
后小号TEP乙10,存储在4℃下过夜样品。在用切片机切割样品之前,请在室温下将波纹管保持在22 °C以防止石蜡软化。
打开切片机。我们建议在手动模式下使用切片机。可以使用电动模式,但是我们没有对其进行测试。用于切割厚度的设置有:3 - 5微米(˚F或多个信息:参见用户手册徕卡的,型号:RM2265 )。
手动从模具中取出组织标本的石蜡块,修剪石蜡块,然后将其固定在切片机的样品夹和固定器中(补充图1H)。此步骤将确保干净且均匀的切割,并形成直的色带,如补充图1K所示。
修剪蜡块拿到面临的刀切片标本的切割面(参考图1我),如果你需要它。
通过使用刷子(捕获部分补充图1K),并把它们在聚-L-赖氨酸处理过的显微镜载片(参考图1L)或8孔ibidi μ-载玻片(补充图1M)不反转的部分,即,位置带电的载玻片或ibidi井底玻璃上面向刀的组织部分一侧(“发光”侧)。
添加的水的液滴betw EEN在滑动件和串行组织切片上滑动或单个ibidi阱(参考图小号1 N和O,小号EE下面注)。
注意:如果使用的ibidi多孔载玻片dSTORM样品制备中,添加0.1%的聚-L-赖氨酸溶液代替水在此阶段。
将载玻片在42 ° C保持3分钟。
[R EMOVE水仔细而不触及削减。残留的任何水滴都会形成气泡,并导致这部分样品的损失。在此阶段,通过以手臂的长度摆动1或2次来手动旋转干燥幻灯片(补充图1P)。
注意:只有在大流行期间才需要戴口罩和手套。
在加热板上于37 °C放置至少干燥过夜(补充图1Q)。载玻片可以在室温下保存至少一个月。使用显微镜载玻片盒进行存放,避免灰尘。
Deparaffining (补充˚F igure 1R) :将最多8个微垂直滑动(或16个滑动背靠背)到250ml科普林玻璃染色缸和浸泡在三个连续的滑动用200ml治疗Histoclear各30分钟,在正常光线条件下的室温下,无需摇晃。然后洗涤该Histoclear -处理microslides与200毫升的使用250ml玻璃的100%乙醇染色罐20分钟。
注意:可以在下一步中的下一个重复使用罐子。不要用水冲洗罐子;只需倒出广口瓶中的溶液即可。
再水化用连续的样品处理使用相同的两个玻璃罐子在前面的步骤中使用,每次15分钟,在100%乙醇,70%乙醇,50%乙醇,25%乙醇,10%乙醇中2F4缓冲器,最后100%2F4缓冲液,在室温下在正常光照条件下,无需摇动。每个步骤使用约200 ml的体积。
注意:可以在下一步中的下一个重复使用该jar。不要用水冲洗罐子;只需倒出广口瓶中的溶液即可。
进行到小号TEPÇ 1 (Immunolabeli NG)尽快,以避免部分的干燥。
笔记:
在小号TEP乙12 ,样品应事先制备并在4保持° ℃过夜。然而,样品也可以是切口(小号TEPS乙-12-乙18)后1个小时小号TEP乙10.在这种情况下,存储在所述样品-20 °下搅拌1小时之前,切割和切割在不到15分钟后从-20 ° C取样
固定在70%乙醇中并在100%乙醇中脱水后,请使用Eppendorf试管存储组织。
 
样品存储断点:完全替换为100%Histoclear后,样品可以在RT下存储几天。但是,Histoclear易挥发,可以溶解石蜡。因此,建议不要超过几个星期。固定后,样品可以在4°C下保存几个月。包埋后,样品可以在低于25°C的温度下保存数年。用切片机切割后,微片上的样品可以在室温下保存数周。


免疫标记
我们提出多色用针对低2F4抗体的免疫染色的一例甲基酯化homogalacturonan结合h的二聚体缔合通过钙离子omogalacturonans ,LM20高甲基酯化homogalacturonan,JIM7用于部分甲基酯化homogalacturonan,CBM 3和CBM 4识别大多结晶和无定形纤维素分别,PDM识别甘露聚糖和LM24识别木葡聚糖。对于所有步骤,即使不使用2F4抗体,也要使用2F4缓冲液代替PBS。它已被证明可以与CMB3和CMB4不同的LM和JIM抗体以及微管抗体(有关可用的LM,JIM和其他抗体以及植物探针的列表,请在此处访问:www.plantprobes.net)正确使用组织切割拟南芥,大米,杨桃和玉米的样品(Yang等人,2016)。重要的是,如果在任何阶段用水或PBS冲洗该缓冲液,您将失去2F4抗体染色。
笔记:
免疫染色之前,可选地,在2F4缓冲液中使用50 mM NH 4 Cl淬灭游离醛基15分钟。用2F4缓冲液洗涤3次,每次3-5分钟。建议执行此步骤,因为样品固定步骤B2中使用的FAA溶液中的任何残留醛基都会与抗体的氨基反应,从而导致非特异性抗体结合。
我们建议按照步骤s C 1 - C5的顺序依次应用所有一抗,以防止竞争和空间位阻效应,尤其是对于位置紧密的表位,例如此处介绍的细胞壁靶标(纤维素,木葡聚糖,果胶和甘露聚糖,图1)。但是,在大多数情况下,同时进行一抗孵育非常有效。
为避免非特异性抗体结合,我们在2F4缓冲液中使用5%的牛奶作为封闭缓冲液。但是,随着时间的流逝,牛奶可能会受到污染。因此,请在不到72小时内完成免疫染色步骤C1-C5 ,并在必要时于4°C进行过夜抗体孵育。在室温(RT)下进行简短的抗体孵育和洗涤步骤。
染色时间表。对于薄组织切片(<5 µm),在室温下2 h一抗孵育时间就足够了。对于较厚的切口和某些抗体,此时间延长至最少3小时。
微波治疗。根据制造商的不同,选定的抗体可能需要微波加热才能激活或加速反应(400瓦时1-2分钟)。此步骤应快速以防止样品沸腾–微波加热后,该方案中提出的抗体均未显示出更高的标记效率。
对于来自Sigma的曲霉果胶酶的酶处理,请用200μ升的果胶酶在温育缓冲液(见ř ecipes)在最终稀释0.1%在室温下10分钟至之前。步骤C 1 (CBM孵育)或步骤C2 (初级抗体孵育)。酶处理后,每次3-5分钟用2F4缓冲液清洗切片3次。当您使用其他果胶裂解酶如Megazyme的E-PLYCJ,检查他们的publica-灰名单的反应条件。
如何制作手工制作的潮湿箱(补充图1U)。将2-3层纸巾放在空的微量移液器盒(10 cm x 14 cm x 9.5 cm)或用于显微镜载玻片托盘的空盒(3 cm x 8.5 cm x 21 cm)的底部,并加入蒸馏水( 〜20 ml),以使纸巾湿润。盖好盒子。用铝箔包裹潮湿的房间。如果您使用共焦成像样品制备微滑梯,确保样品的的MicroSlide是不是与接触湿纸巾。为此,我们建议使用显微镜载玻片托盘。
在每一步中,我们使用200微升理论值的ë每盖玻片溶液含有抗体的(补充˚F igure 1S),而我们使用50或70-100微升的含有抗体的溶液每8孔的单个孔中ibidi滑动(补充˚F igure 1T,小号EE人所以步骤C 8 )。用于洗涤,我们使用大约500微升每盖玻片或200微升每单井2F4的用5%乳缓冲液。将切片放置在液滴的中心。试剂(抗体)在液滴上浓缩(一种称为咖啡渍的过程)。在这里,我们提出以下最佳顺序抗体染色顺序。可选地,在抗体或试剂孵育之前,执行步骤C 1或C 2,将您的样品在封闭缓冲液中孵育30分钟。然后直接去小号TEP Ç 1 。
 
纤维素结合分子(CBM3或CBM4)孵育:在封闭缓冲液中稀释2/100体积/体积(v / v)CBM3 / 4试剂,然后添加到样品中。孵育2在室温或4℃过夜ħ ℃下在潮湿的腔室(补充˚F igure 1U) 。除去CBM3溶液后,用封闭缓冲液洗涤3次,每次5分钟。如果隔天在4°C下进行过夜孵育,则在清洗步骤前至少30分钟将潮湿的样品室从冰箱中取出。
第一次一级抗体孵育:在封闭缓冲液中稀释20/100 v / v 2F4抗体,然后添加到样品中。在室温下孵育2小时,或在潮湿箱中于4 °C过夜。除去2F4溶液后,使用封闭缓冲液洗涤3次,每次5分钟。如果隔天在4°C下进行过夜孵育,则在清洗步骤前至少30分钟将潮湿的样品室从冰箱中取出。
第二次一抗孵育:在封闭缓冲液中稀释10/100 v / v LM20抗体,然后添加到样品中。在室温下孵育2小时,或在潮湿箱中于4 °C过夜。去除LM20溶液后,使用封闭缓冲液洗涤3次,每次5分钟。如果隔天在4°C下进行过夜孵育,则在清洗步骤前至少30分钟将样品从冰箱中取出。
第三次初级抗体孵育:在封闭缓冲液中稀释1/100 v / v兔或鸡抗组氨酸标签抗体,然后添加到样品中。在室温下孵育2小时,或在潮湿箱中于4 °C过夜。除去抗体溶液后,使用封闭缓冲液洗涤3次,每次5分钟。如果隔天在4°C下进行过夜孵育,则在清洗步骤前至少30分钟将潮湿的样品室从冰箱中取出。
二级抗体孵育:在与二级抗体混合物相同的封闭缓冲液中稀释所有三种二级抗体1/100 v / v 。在室温下孵育2小时,或在潮湿箱中于4 °C过夜。在黑暗中孵育二抗,以避免光致漂白。为此,用铝箔纸包裹加湿室。除去后次级抗体-混合物溶液,洗3次,用封闭缓冲液5分钟。如果隔天在4°C下进行过夜孵育,则在清洗步骤前至少30分钟将潮湿的样品室从冰箱中取出。Ť他的第二抗体温育也可以顺序进行(参见Ť故障排除小节),虽然在的情况下˚F igure的1〜4 ,同时加入所有的次级抗体。
笔记:
步骤C 1 - C 5 :在加湿室中执行所有抗体孵育步骤。
使用另一种物种(例如兔,豚鼠,马和人)的抗体,可以将该协议扩展到三个以上的靶标。但是,在共聚焦显微镜上对四种以上的颜色进行成像需要附加的激光线(> 700 nm)或光谱分解步骤。
使负控制图像而不1日,2次,3种p rimary抗体像图1 A(只有。步骤C 5至少在第一个试验的情况下,用第二抗体混合物。
对于dSTORM成像,从步骤B14开始,我们建议使用多孔Ibidi玻璃底μ玻片,例如8孔玻片(Ibidi ),可以轻松地将其插入并固定在许多显微镜载物台上。多孔载玻片方便了几种成像条件的准备,减少了试剂用量,提高了半定量分析免疫染色的均一性。对于8孔载玻片Ibidi,我们推荐使用70-100微升每孔的试剂在步骤小号Ç 1 - C ^ 5的免疫标记。
 
后免疫固定。
此步骤对于dSTORM纳米显微镜样品很重要,对于使用固定介质进行共焦成像的样品则不需要。由于与共聚焦显微镜样品相反,dSTORM样品未安装在安装介质中,因此它会降低抗体复合物的热运动并减慢抗体从表位的解离。
温育样品10分钟在3.7%甲醛(小号EE配方4)在10适量稀释X 2F4缓冲器,并在室温蒸馏水。为此,将200 µl的3.7%甲醛溶液滴在ibidi多孔载玻片内部的样品上。在通风橱中执行此步骤,并戴上丁腈手套。
用3 00 µl 2F4缓冲液洗涤3次,每次3分钟。在通风橱中执行此步骤。
猝灭70甲醛μL下降的15分钟的50mM氯化铵。此步骤可降低在成像过程中处理样品时与甲醛接触皮肤的风险。稀氯化铵在2F4缓冲液(见ř ecipes)。温育后,洗涤简要3次后3 00微升液滴的2F4缓冲器。您也可以使用其他含有胺基的醛猝灭剂,例如2F4缓冲液中的50 mM甘氨酸。
样品安装和存放。仅用于共焦成像。
注意:请勿为dSTORM样品制备执行此步骤。
要延长样品的使用寿命,请用盖玻片将载玻片安装在含有抗褪色剂的固定介质中。我们建议的ProLong Gold抗淬灭封固(赛默飞世尔科技)(补充˚F igure 1V) 。为了防止气泡的形成,放置在盖玻片封固的液滴,并轻轻将其放下上用微型镊子滑动(补充˚F igure 1W-AA) 。密封用指甲油盖玻片,以防止干燥  (补充˚F igure 1AB)和把密封的微滑动到纸板滑动托盘(补充˚F igure1AC)和存储在黑暗中在4 ℃。
样品储存。仅用于dSTORM成像。
完成后小号TEPS Ç 6 ,一个DD 500微升的2F4缓冲到各8个孔,密封用石蜡膜中,包裹物在铝箔上,并保持在4潮湿室℃,如在呈现补充图1AD和1 AE 。
注意:您可以执行此操作以进行共焦成像,但是您无法将样品装入抗褪色封固剂中;因此,不建议这样做。
选择荧光染料。
对于共聚焦成像,我们建议使用明亮且耐光的染料,例如Alexa Fluor,ATTO和CF系列。对于dSTORM成像,仅某些染料有效。我们建议使用ATTO488,CF568和Alexa647染料。对于dSTORM纳米显微镜成像,我们建议使用F(ab')2二抗片段或纳米抗体,或者在可能的情况下,将一抗直接与目标荧光团偶联。对于密集包装的表位,我们建议使用单个标记的二抗。
 
故障排除
高背景:降低二抗浓度。优良作法是进行一抗和二抗的滴定。例如,如果建议的抗体稀释度是1:200,则滴定测试的一个好的起点是1:50、1:100、1:200、1:400和1:800稀释度。免疫染色之前,在2F4缓冲液中使用50 mM NH 4 Cl淬灭游离醛基15分钟。在已经产生第二抗体的封闭缓冲液中使用血清,例如,山羊血清中的山羊血清。在S tep C 1或C2之前,将抗体在4°C下孵育过夜。
  预期不到的高度共定位:对所有第二抗体使用顺序染色。如果可能,对所有第二抗体使用相同的宿主物种(例如,来自山羊的一组抗大鼠抗体,抗兔抗体和抗小鼠抗体等)。为避免脱靶结合,请使用高度交叉吸附的二抗。我们建议准备仅执行步骤C5的测试样品,这是应用第二抗体而没有第一抗体的情况。
  低信号:增加一抗浓度和/或孵育时间。尝试使用新一批的一抗和二抗。更改第二抗体(共轭染料,宿主物种或同一类型抗体的生产者)。增加二次抗体浓度(进行的滴定-试验在2,4,和6倍增加的抗体稀释液)和/或温育时间(在2小时,4小时,6小时进行测试)。检查2F4缓冲液的pH。如果pH值不为8.0,则进行调整。
 
共焦成像技巧
1)复染      
对于用共聚焦显微镜对细胞壁成分进行半定量分析,请比较来自相同实验制备物的样品。如果可能,请考虑使用激光扫描共聚焦显微镜的第三或第四通道观察复染样品,如果预计复染强度在不同样品条件下不会改变。对于细胞壁染色,请使用例如钙氟荧光白染色剂。添加200微升的卡尔科弗卢尔白色染色后小号TEP Ç 5以5mM浓度在2F4缓冲器和孵育5分钟在室温下。洗涤两次简要地用500微升的2F4缓冲区之前施加的抗褪色封固介质的样品(步骤C 7 )。复染可用于将强度数据规格化为大小,例如面积或单元壁厚度。准备每个实验准备要比较的所有条件。
2)显微镜操作      
  成像前1h打开激光器。在成像期间,请确保不要饱和检测器上的信号以进行下游定量。在整个实验过程中保持激光功率和检测器增益相同,并且在可比较实验之间保持所有其他设置不变。此外,为了避免在最终拍摄之前优化图像质量的过程中出现光致褪色,请考虑使用科学兴趣较小但幻灯片上存在相似信号水平的组织样本或区域,1)找到目标点和焦点在明亮的领域。如果在显微镜上安装了DIC,则微分干涉对比图像(DIC)可以更方便地聚焦图像。2)在最终目标点附近的位置优化图像质量。3)在优化图像质量期间,请频繁停止扫描。4)作为共聚焦激光扫描模式的初始设置,激光功率的1%至2%是聚焦样品的良好起点。5)建议以更快或最大的扫描速度(任意水平的扫描速度:在Zeiss LSM 710中为> 9)来关注初始条件(ZEN软件的Live按钮在Zeiss LSM 710中很方便)。
注意:根据Zeiss当地办事处的说法,LSM710有15种任意级别的扫描速度,最大级别“ 15”时的绝对速度为8帧/秒。当执行定量成像,由于光漂白,从未采取最终图像的同一区域的两倍。因此,在特定方向(从右到左,从前到后)移动微型载片的XYZ载物台,以避免观察到同一点。为了进行可靠的定量,需要进行2-3个独立的实验(独立的技术重复)并进行比较。执行先导实验以确定通用参数,例如增益和像素停留时间,并消除/最小化任何光漂白。对所有通道进行顺序成像,始终从最长的波长开始,到最短的波长结束。


数据分析
 
图1显示了不同细胞壁表位的双色和三色IHC,代表了细胞壁多糖三个不同家族的成员:果胶(2F4,LM20和JIM7),纤维素(CBM4,CBM3)和半纤维素(PDM和LM24) )在杨桃叶轴的横切面(补充图2和表1 )。
  比较图1的A和C,在高HG计数区域和高HG计数较低的组织中,纤维素染色(CBM3和CBM4 ,参见表1 )较弱。这可能表明纤维素和果胶占据了排他性的壁室。然而,酶的果胶提取转ealed高得多的纤维素检测水平与CBM4 (比较图小号1C和1E)。这表明,在完整的细胞壁中,大多数纤维素表位被果胶所掩盖。如果不进行果胶提取,则剩余的纤维素染色可能与木葡聚糖相对应,这可由CBM探针部分检测到(参见PlantProbes CMB3试剂说明,Hernandez-Gomez等,2015)。实际上,图1D显示针对木葡聚糖的CBM3和LM24抗体在壁的子集中呈现出低程度的重叠。
 
图1.杨桃(杨桃)叶片叶轴上不同细胞壁表位的多色共聚焦图像。A.仅用二抗染色的对照图像。B. 2F4,CBM4和LM20和(C)2F4,CBM4和JIM7表位的三重染色。D.双染色CBM3和LM24的。E.酶法提取果胶后,对CBM4和PDM进行双重染色。所有图像均使用相同的显微镜设置获取(建议的设置请参见表2 )。最后一栏代表切割的透射图像。图像在斐济可视化。比例尺小号,100微米。


表1.图1-4中使用的第一抗体,CBM试剂和第二抗体稀释液
 


表2.使用共焦激光扫描显微镜对三重免疫染色和钙氟荧光白复染色的建议发射滤光器带宽,优化后可将光谱串扰减至最小。有关dSTORM滤波器组的详细信息,请参阅Haas等。(2020年)
 
  图2显示了拟南芥子叶L2层(表皮下的一层)中不同果胶种类的3D dSTORM纳米显微镜成像,其纵向剖面在补充图2E中进行了描述。在图2 A和2 B,低甲基化(2F4),高甲基化(LM20),并且观察到部分地甲基化(JIM7)果胶的典型小区边缘染色(见表1) 。三维dSTORM图像显示,该染色细胞壁(间层)内弱,大多局限于面临的细胞质单元壁边缘-t他的亮点IHC的一般最大限制之一:表位可访问性和渗透性抗体。然而,在该具有相似的空间和化学分辨率(可得技术光例如,IEM ),3D dSTORM提供总体较高的标记和检测的密度,并在中间薄片检测表位的数目仍然是3D更高dSTORM比IEM (科斯格罗夫和安德森(Anderson),2020年。绕过此限制的一种可能方法是从细胞壁中去除一种聚合物(例如,被重组蛋白酶K降解的结构蛋白)以增加抗体渗透。但是,这可能会引起非特定的壁扰动。针对细胞壁靶标的小型纳米抗体的开发将在将来帮助解决这些局限性。相关超分辨率光学和电子显微镜的进步可能有助于提高性能,并消除3D dSTORM和EM的局限性。
 
图2.在拟南芥子叶的L2(疏皮细胞)层的三细胞交界处的同型半乳糖醛酸的两色3D dSTORM成像。两色散点图显示了定位的(A)JIM7(绿色)和2F4(紫色)以及(B)LM20(绿色)和2F4(紫色)表位的3D坐标。图像插图显示了低分辨率倾斜照明图像,并在散点图中显示了红色正方形轮廓区域。2D –二维顶视图(XY)和3D – 3D倾斜视图,其中Z轴垂直于常规图像平面。橙色线表示在下面的面板中放大的细胞壁区域。图像比例尺,4 µm。使用GrafeoV.2可视化数据。
 
  图3示出了3D dSTORM纳米显微镜中的表皮层的不同果胶物质的检测的拟南芥子叶具有纵向部分中补充图2G所述。在这两种˚F igure小号3A和3 B,2F4和LM20的表位形成的背斜,但不是平周壁染色的丝状图案,先前描述为HG纳米丝(哈斯等人,2020) 。此纳米丝四级结构是具有挑战性的果胶的规范视图中Muro的架构,为无定形凝胶状基质。这些出乎意料的结果引起两个问题:(1)丝状图案将果胶表示为纤维素微纤维之间的间隔物,或(2)果胶抗体无法穿透由纤维素微纤维形成的凹槽之间(Cosgrove and Anderson,2020)。为了解决这些问题,我们在子叶的背壁上标记了部分甲基化的HG表位(JIM7),图3A。与LM20和2F4 HG表位相比,J IM 7呈现了更为广泛和统一的分布模式。首先,与LM20 / 2F4丝状分布相比,JIM7表位的扩散分布表明果胶甲基化模式与其定位有关。其次,这意味着LM20和2F4的HG纳米丝是可能的,这不是由于纤维素微纤维形成的凹槽中的表位不可及,因为JIM7表位可以定位在此处,图3A。此外,在未事先提取果胶的情况下,对未成熟叶片第6个节点(补充图2C和F中的纵向截面)的玉米表皮细胞中的纤维素(CBM3)和HG(2F4)进行了双重染色,结果表明2F4纳米丝之间没有空隙纤维素没有被填充,并且没有果胶提取的纤维素染色的整体水平非常低,图3C。
 
图3.双色3 d dSTORM在的波瓣Homogalacturonan的成像拟南芥子叶路面细胞和玉米叶路面细胞。二维彩色散点图示出局部(A)JIM7(绿色)和2F4(紫色)的三维坐标,和(B)LM20(绿色)和2F4(紫色)的表位在拟南芥子叶。图像插图显示了3D dSTORM散点图中显示的区域的低分辨率荧光图像。橙色线表示在下面的面板中放大的细胞壁区域。(C)两色散点图,显示了玉米叶片中局部2F4(紫)和CBM3(橙色)表位的3D坐标。比例尺,1 µm。使用GrafeoV.2可视化数据。
 
  图4示出了双色3D dSTORM的成像拟南芥原基和分生组织朝向纤维素(CBM3或CBM4)和(杂)甘露聚糖(PDM)和与现有果胶提取具有1cm的纵向截面的的花序(补充˚F igure小号2B和2 D ,表1 )。比较面板A和B显示,检测到主要为结晶性纤维素的CBM3形成了离散的细丝分布,而与CBM4相比,CBM3标记了无定形纤维素。甘露聚糖的染色在不同的细胞壁中显示出不同的丰度:从大部分不包含在纤维素中的扩展壁部分(图4A)到点缀在纤维素纳米原纤维的染色装饰末端(图4B)。
 
 
图4.两色dSTORM在纤维素的成像(CBM3 / 4)和(杂),甘露聚糖(PDM)拟南芥原基,和分生组织。佩奇吨插件瓦特ERE酶促与萃取果胶酶之前的免疫染色协议处理(小号TEP Ç 1 )。两色散点图显示了分生组织中局部(A)CBM4(蓝色)和PDM(橙色)以及(B)原基中CBM3(蓝色)和PDM(橙色)表位的3D坐标。曲线左侧的插图显示了一个低分辨率的荧光图像,并在散点图中显示了红色正方形轮廓区域。比例尺,1 µm。使用GrafeoV.2可视化数据。
 
结论
IHC是研究细胞壁化学中亚细胞变化的有力工具。但是,它有几个限制。由于光谱串扰和抗体种类的数量,可以同时探索的表位数量有限(<4个)。此外,只能检测到具有明确抗体的表位。此外,除非细胞壁成分之一被降解以帮助穿透,否则抗体对内部细胞壁成分的可及性受到限制。同样,用于IHC的样品制备可能会导致组织变形;因此,空间尺度的测量和拓扑可能不是绝对的。尽管如此,多色IHC与新颖的光学纳米技术相结合,为生物分子的复杂空间组织提供了前所未有的见解。结合超分辨率成像,多色IHC现在正在改变我们对细胞壁结构和植物生长的看法。细胞壁介导植物细胞与环境的相互作用,因此我们希望该技术将帮助我们了解植物免疫力,果实成熟,植物与微生物的相互作用,植物生长和产量。荧光探针,单分子技术和数据分析要使常数改进,3D dSTORM有巨大的潜力,以精益求精的分子组件和它们的功能我们的知识(克莱恩ê牛逼人。2014年,金等人。,2019; Gwosch等。,2020;张等人,2020) 。因此,“与流行的看法相反,眼见为实。在这里,信念就停止了,因为不再需要信念了。” ,金字塔的Terry Prattchet
 
菜谱
 
2F4缓冲液(T / Ca / S缓冲液终浓度)
Ť ø稀所有的抗体,制备封闭缓冲液和洗涤缓冲液(http://www.plantprobes.net/pp_2F4.pdf)。
Tris-HCl 20 mM pH 8.2
氯化钙2 0.5 mM
氯化钠150 mM
最终pH应为8.0
˚F ormaldehyde一个lcohol一个cetic酸FAA解决方案
50%乙醇
10%醋酸
甲醛3.7%
百分比表示FAA溶液中的最终浓度
在2F4缓冲液中稀释的1 M氯化铵溶液
将53.489 g氯化铵粉末混入1 L 2F4缓冲液中
调节pH至8.0
甲醛稀释于10x 2F4缓冲液中
混合1体积的37%甲醛溶液,1体积的10 x 2F4溶液和8体积的蒸馏水。
在通风橱下表演
用于果胶裂解酶孵育的柠檬酸-磷酸盐缓冲液(pH 4.8,Stoll和Blanchard,1990年)
0.2 M Na 2 HPO 4 · 7H 2 O
将53.65 g溶于MilliQ水中,制成1 L溶液A
0.1 M柠檬酸
将19.21 g溶于MilliQ水中,制成1 L溶液
混合24.8 ml的溶液A和25.2 ml的溶液B并稀释至100 ml,pH值为4.8
如果需要,检查pH值
 
致谢
 
这项工作提出了扩展协议,该协议已在Haas等人的文章中发表。,2020年。dSTORM在MRC分子生物学MRC实验室(剑桥)进行,我们感谢Nick Barry和Jonathan Howe的支持。我们为赫尔曼·霍夫特(HermanHöfte)提供帮助,帮助他们进行筹款和结果讨论。
  资金:AP获得了法国国家研究局(ANR)GoodVibration ANR-17-CE13-0007和欧盟的支持,这是通过授予AgreenSkills +奖学金(根据授权协议号201310)。IJPB受益于Saclay Plant Sciences-SPS(ANR-17-EUR-0007)的支持。塞恩斯伯里实验室的显微镜设施得到盖茨比慈善基金会的支持。这项工作得益于IJPB的植物观测台技术平台的支持。
 
利益争夺
 
作者宣称没有利益冲突。
 
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Haas, K. T., Rivière, M., Wightman, R. and Peaucelle, A. (2020). Multitarget Immunohistochemistry for Confocal and Super-resolution Imaging of Plant Cell Wall Polysaccharides. Bio-protocol 10(19): e3783. DOI: 10.21769/BioProtoc.3783.
  2. Haas, K. T., Wightman, R., Meyerowitz, E. M. and Peaucelle, A. (2020). Pectin homogalacturonan nanofilament expansion drives morphogenesis in plant epidermal cells. Science 367(6481): 1003-1007.
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