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

Overview

In this video, we describe the Maule staining technique for staining syringyl lignin present in the specialized plant cells. This staining technique helps to characterize the lignin distribution in the xylem and interfascicular fibers.

Protocol

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.

Representative Results

Figure 1. Mäule staining of A. thaliana stem cross-sections. Mäule staining (red color) of a wild type A. thaliana stem section showing the normal lignin deposition in the walls of interfascicular fibers and xylem cells (A-E). The positions of the epidermis and cortex (Ep), interfascicular fibers (Fi), pith (Pi), and xylem (Xy) are indicated. Magnifications: Panel A: 5X; Panel B: 10X; Panel C: 20X; Panels D and E: 40X. Bar = 100 µm.

Materials

Name	Company	Catalog Number	Comments
Agarose	EMD	MERC2125	CAS Number: 9012-36-6
Hydrochloric acid	EMD	HX0603-75	CAS Number: 7647-01-0
Vibratome	Leica	Leica Vibrating blade microtome VT1000S	http://www.leicabiosystems.com/ products/sectioning/vibratingblade-microtomes/details/product/ leica-vt1000-s/
Razor	American Safety razor company	Item # 60-0139-0000	Stainless Steel Double Edge Blade (Personna Super)
Screw Cap Microcentrifuge Tubes (2 ml)	VWR	16466-044
Microcentrifuge Tubes (0.6 ml)	Axygen Scientific	MCT-060-C
Tissue adhesive	Ted Pella Inc	10033	Store at 4 °C or 20 °C for 3 months or longer storage
Camera with CCD chip with no mechanical shutter	Hamamatsu	C4742-95
Camera software	Molecular Devices	MetaMorph version 7.7.0.0
Imagining anaylsis	Adobe	Photoshop CS4
Micro Cover Glasses, Square, No. 1	VWR	48366-067	22 x 22 mm (7/8 x 7/8")-Cover glasses are corrosion-resistant
Frosted Micro Slides, 1 mm	VWR	48312-003	75 x 25 mm - 1 mm
Ammonium hydroxide	EMD	AX1303-6	CAS Number: 1336-21-6
Potassium permanganate	Sigma	223468	CAS Number 7722-64-7