Maule Staining for Lignin: A Technique to Characterize Lignin Distribution in Arabidopsis Thaliana Stem Sections

1. Mäule Staining

1. Dissolve 0.2 g of potassium permanganate in 40 ml distilled water to prepare a 0.5% potassium permanganate solution. Store in a dark bottle at room temperature for a maximum of 7 days.
   1. Prepare a 3.7% HCl solution by adding 1 ml concentrated HCl (37 N) to 9 ml distilled water (1:10). Prepare a fresh 3.7% HCl solution on the day of the experiment. Make sure that the 37 N HCl stock solution being used is not too old. Concentrated ammonium hydroxide solution (14.8 M) should be stored at 4 °C to prevent the molarity of the saturated solution from decreasing.
2. Transfer stem sections to a 2.0 ml microcentrifuge tube. Add 1 ml of the 0.5% potassium permanganate solution to the tube containing the sections.
   1. Pipette the 0.5% potassium permanganate solution up and down gently, preferably without disturbing the sections. Incubate for 2 min and repeat the pipetting. Let the microcentrifuge tube stand until all the sections settle down.
      NOTE: Be careful not to pipette the sections into the pipette tip as the sections may get damaged. It may be hard to see the stem sections as the solution is dark, so keep the tube with the sections on test tube rack and allow them to settle for 2 min.
   2. Using a 1 ml pipette, draw out 700 µl of 0.5% potassium permanganate solution. Add 700 µl of distilled water to rinse out the potassium permanganate solution. Repeat 3-4x or until the water solution stays clear.
   3. Discard the water. Quickly add 1 ml of 3% HCl until the deep brown color is discharged from the sections. This may happen within 3-5 min or may require 2 washes of 5 min each. Pipette out all the 3% HCl solution and immediately proceed to the next step.
   4. Add 1 ml of concentrated ammonium hydroxide solution. Use a 1 ml pipette with the tip cut in such a way that the sections can be pipetted out easily without damage. Gently pipette sections into the tip and onto a microscope slide. Cover the sections with a coverslip. Observe the sections under bright-field lighting. (Figure 1)
      CAUTION: Because ammonium hydroxide is extremely corrosive, it requires extra care and attention to avoid causing corrosion to the stage of the microscope or lens.
      NOTE: The ammonium hydroxide fumes can fog the coverslip, making it difficult to observe the sample. Therefore, be extra careful before adding the coverslip. The solution dries up in 5-10 min, so the imaging must be completed within that period of time.