Aniline blue staining of quanatification of plasmodesmal callose

  1. Grow Arabidopsis plants in short days conditions (10h of light) at 20oC for five weeks
  2. When plants are 5-6 weeks old, water the plants and cover them with a lid the day before to make the infiltration easier and reduce the “wounding effect”
  3. Infiltrate the 8th leaf with a 1 mL syringe containing01% aniline blue (Sigma-Aldrich #415049, in PBS buffer, pH=7) See figure for leaf numbering (Farmer et al., Nature (2013)
  4. Preparing the microscope slide for confocal imaging
    1. Spray the middle part of the microscope slide with medical adhesive spray and let it dry, when it is almost totally dry but still sticky, it is ready to use
    2. Cut the leaf in halves by removing the mid rib and put the halves onto the prepared microscope slide with abaxial side up. Carefully flatten the leaf halves, avoid damaging the sample
    3. Cover the sample with water and coverslip
  5. The settings for Leica SP5 confocal microscope:
    1. 405nm laser, UV (15%), Spectral range (DAPI): 430nm-550nm
    2. resolution (big depth): 16 bits
    3. lens: 63X water if possible (oil lens can modify Z-axis)
    4. signal: PMT (or HyD & photon counting, Line accum: 4, expand display)
    5. Acquisition settings;
      Acquisition mode: XYZ
      Format: 1024×1024
      Speed: 400Hz
      Zoom factor: 1
      Line average: 4
      Frame average: 1
      Pinhole: set to the peak of aniline blue
      Z-stack steps: 3 μm: set to optical thickness! (between ½ and 1 optical thickness)
      Adjust gain for the sample with the highest intensity (biggest callose deposition) to avoid saturation
  1. While imaging, keep in mind that the aniline blue can be bleached out easily with UV light! While screen for a good area to image always use brightfield light and just shortly switch on the UV light before imaging
  2. Collect 5 image-series for each half leaf. Image 3-3 leaves per condition and repeat the experiment. Always use the same settings in the independent experiments
  3. Compare the intensity and voxel size of PD callose in the samples with different conditions using automated image analysis