Materials and Reagents

Square dishes 120 × 120 × 17 mm (Greiner Bio-One GmbH, catalog number: 688102 )
Flat-bottom 6-, 12-, and 24-well plates with lid (Stemcell Technology, catalog number: 100-0096 )
Adhesive or any available tape
2 ml Eppendorf tubes
Falcon tubes 15 or 50 ml (Stemcell Technology, catalog numbers: 38010, 100-0090; 38009, 100-0092 )
Aluminum foil
Parafilm “M” (Merck, catalog number: P7543-1EA )
SuperFrost microscope slides (Thermo Scientific, catalog number: 12362098 )
Coverslips (Thermo Scientific, catalog number: 12393138)
Note: Material needed for the protocol is illustrated in Figure 1.
Chambered cover glass (VWR, chamber slides-one chamber, Nunc^TM Lab-Tek^TM, catalog number: 734-2056 )
Arabidopsis seeds
GROWTH CONDITIONs: Sow surface-sterilized Arabidopsis seeds on half-strength MS agar square plates (45.5 ml). Stratify plates for 2 days at 4 °C in the dark and then transfer to growth chambers under a 16-h-light/8-h-dark photoperiod at 21 °C. Allow seedlings to grow on vertically-oriented plates for 5 days.
Note: 5-day old seedlings are the standard age of seedlings in our experimental setups. The age of seedlings can be adapted according to experiments.
Milli-Q Water (H₂O)
Sucrose (VWR International, catalog number: 27483.294 )
Murashige and Skoog basal salt mixture (MS salts) (Duchefa Biochemie, catalog number: M0221.0050 )
2-[N-morpholino] ethanesulfonic acid (MES) (Duchefa Biochemie, catalog number: M1503.0100 )
Agar (Lab M, catalog number: MC029 )
Potassium hydroxide (KOH) (Merck KGaA, catalog number: 1.05021.1000 )
Ethanol (EtOH) (Sigma-Aldrich, catalog number: 32221-2.5L )
Lactic acid (Honeywell, Fluka^TM, catalog number: 15636730 )
Fluorol Yellow 088 (Santa Cruz Biotechnology, catalog number: 81-37-8 )
Aniline blue (Sigma-Aldrich, catalog number: 66687-07-8), 0.5% in MilliQ Water
Seed sterilization with ethanol (see Recipes)
Half-strength MS medium (see Recipes)
0.03% Fluorol Yellow 088 solution (see Recipes)
0.5% Aniline blue solution (see Recipes)

Figure 1. Required material and equipment. A. Five-day-old Arabidopsis seedlings. Scale bar: 1 cm. B. Materials needed to stain samples. C. Dissolving Fluorol Yellow 088 in lactic acid in a 50 ml Falcon tube attached to a vortex mixer with adhesive tape. D. Dissolved Fluorol Yellow (left) and aniline blue (right) solutions.

Equipment

Growth chamber to grow plant materia (Percival, CLF, Plant Climatics, AR75-L3 )
Vortex mixer (Instrumentfirman Labora, model: Vortex-Genie 2 )
Water bath (Fisher Scientific)
Soft stainless steel tweezers (3B DISSECTION SOFT TWEEZERS, LABWORLD)
Inverted confocal microscope (Zeiss, model: LSM880 )
Note: A fully motorized X, Y, Z scanning stage is required to perform tile scan image acquisition experiments.
Objectives: 10× or 20× (suitable for monitoring the whole specimen or root, e.g., Figure 3C). For higher resolution images, a higher magnification objective, such as 40× or 63×, is required (Figure 3D)
Fluorescence signal detection system for GFP and other fluorescent reporters (Shaner et al., 2007)

Software

Software operating the confocal microscope
ImageJ (Abràmoff et al., 2004)
Microsoft Excel
Prism (GraphPad)

Procedure

Sample staining
1. Add 15-20 seedlings into each well containing 2 ml of Fluorol Yellow 088 (FY088) (e.g., 12-well plate, depending on the number of samples) (Figure 2A).
2. Close the lid and seal the plate with parafilm. Place the plate in a water bath on an aluminum foil raft at 70 °C for 20 min (Figures 2B-2D).
  Note: Bend the edges of the aluminum foil to prevent sinking. During the incubation time in the FY088 solution, prepare the 0.5% aniline blue solution and keep in the dark at room temperature until use.
3. Transfer the seedlings from the FY088 solution to a new 12-well plate containing 2 ml water and incubate for 1 min at room temperature (Figure 2E).
4. Remove water by pipetting and add 2 ml of 0.5% aniline blue solution to each sample (Figure 2F). Incubate for 20 min in the dark at room temperature.
5. After incubation in aniline blue, move the seedlings to a new -, 12-, and 24-well plates containing 2-3 ml water (Figure 2G) and incubate for 10 min in the dark.
  
  Figure 2. Sample Staining. A. Twelve-well plate containing Arabidopsis seedlings in 2 ml FY088 solution. B. Plate sealed with parafilm. C. Sealed well plate placed on an aluminum foil raft. D. Samples placed into a water bath at 70 °C for 20 min. E. Seedlings washed in 2 ml of water. F. Well plate containing 2 ml aniline blue solution with seedlings. G. Wash aniline blue in 2 ml water. H. Preparation of microscope slide with parafilm strips to make a “spacer”.
Sample preparation/mounting
1. Cut two strips of parafilm and wrap 2-3 times around both ends of the microscope slide (Figures 2H and 3A).
2. Add a few drops of water (30-50 μl) in the center of the slide between two parafilm strips. Position the seedlings onto this liquid using tweezers and cover with a cover glass slide (Figures 3A and 3B).
3. For instructions on using a one-well chambered cover glass and positioning the seedlings, please follow the protocol described previously: Marhavý and Benková, (2015).
Sample imaging
1. Prepare an inverted confocal microscope for use; set lasers (for GFP: 488 nm excitation, emission 500-550 nm) and objectives.
  Note: For a broad overview of suberin localization in the root, we recommend performing tile scan imaging using either a 10× or 20× objective (in dry or water-immersion mode). For higher resolution images, we recommend using a 40× or 63× objective immersed in water or oil.
  Parameters for image capture on a Zeiss LSM 880 shown in Figure 3C: Image size – x: 21793.72 µm, y: 18702.12 µm; Scaling X – 1510 µm, Scaling Y – 1510 µm; Tile scan – tiles1, overlap in percent: 20.0, tiling mode: rectangular grid (stitched image); scan mode – plane, zoom 1.1; Pixel dwell – 0.85 µs; master gain – 482; digital gain – 1.5; digital offset – 0.00; filters – SP 555; beam splitters – MBS: MBS 488 nm, MBS_InVis: plate, FW1: Rear.
  Note: Parameters should be adjusted according to the specimen used.
2. Mount a glass slide or chamber with seedlings and place on the stage of the confocal microscope.
3. Activate the position list and tile scan (25 × 34 tiles) with 20% overlap between images.
  Notes:
  1. Adjust the number of tiles according to the approximate scanning area. Alternatively, activate automatic stitching (this can also be done later using the confocal software).
  2. Parameters should be adjusted according to the size of the samples.
4. Find the root closest to the central point of the scanning area and mark the position.
5. Deactivate the position and start scanning.
  Notes:
  1. With the above-mentioned parameters, it will take approximately 15 min to obtain the final image. This provides enough time to prepare the next sample, as described above.
  2. If the laboratory does not have suitable confocal microscope, images can be taken by a fully motorized Fluorescence stereo microscope with a tile scan option.
6. Export confocal images in TIFF or JPEG format. Open images in ImageJ or other software and analyze your images (Figures 3C, 3D).
  Note: Be exact in terms of both the timing and procedure for each step to ensure reproducibility of your results.
  
  Figure 3. Sample preparation and imaging. A. A 30 μl drop of water placed on the microscope slide between two parafilm strips. B. Place 10-15 seedlings onto each slide and cover with a cover slip. C. Tile scan of stained Arabidopsis seedlings. Yellow fluorescence indicates suberin deposition in the root. The white frame surrounds the area that is expanded in D for a detailed image of suberin lamellae. Scale bar: 1 mm. D. Detailed image of suberin lamellae in endodermal root cells. Scale bar: 100 μm.

Data analysis

Open image in ImageJ Set scale; Known distance; Unit of length (mm or cm); click/mark Global and press OK. Go to ImageJ, select drawing line (segmented or freehand line) and draw a line along the root, from base to tip, press “M” to measure root length. Repeat the procedure to measure the length of the suberized region of the root. Copy the results to Excel or Prisma GraphPad for further processing (for example, to calculate suberin occupancy in the root).

Note: We do not recommend analyzing FY fluorescence intensity, since this can be influenced by discrepancies in staining time, washing, and/or imaging.

Recipes

Seed sterilization with ethanol
Transfer seeds to 2 ml Eppendorf tubes (maximum volume of seeds to be sterilized per tube should not exceed 3 mm from the bottom of the tube)
Add 1 ml 70% EtOH, shake for 5 s and allow seeds to settle for 10 min
Dry seeds under the clean bench
Half-strength MS medium (1 L)
Combine 10 g sucrose, 2.3 g MS salts, and 0.5 g MES, adjust the pH to 5.9 with KOH, add 8 g agar and fill with ddH₂O to 1 L
Autoclave medium for 20-30 min, and allow to cool to 55 °C before pouring plates
0.03% Fluorol Yellow 088 solution
Fluorol Yellow 088 in lactic acid at room temperature (Figures 1C, 1D) must be freshly prepared before each experiment
Note: To speed up dissolving FY 088 in lactic acid, attach a 50 ml Falcon tube to a vortex mixer with adhesive tape and vortex for 10-15 min (Figure 1C).
0.5% Aniline blue solution
Aniline blue in Milli-Q Water
Store in a dark place at room temperature

Acknowledgments

We are thankful to Niko Geldner for all the discussion and feedback to develop this protocol. This work was supported by the Swedish research Council a Starting grant to Peter Marhavý. Shahid Siddique was supported by a grant from the United States Department of Agriculture (project no. CA-D-ENM-2562-RR).

The protocol was improved based on previously published work of Lux et al. (2005).

Competing interests

There are no conflicts of interest or competing interest.

References

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