Wiesner Staining for Visualizing Lignin: A Technique to Stain Lignin Deposits Present in Stems of Arabidopsis Thaliana

Abstract

Source: Pradhan Mitra, P. et al., Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements. J. Vis. Exp. (2014).

In this video, we describe the Wiesner staining technique for staining the lignin present in specialized plant cells. This staining technique helps to visualize the lignified cell walls in Arabidopsis thaliana stems and helps to quantify the lignification in the cell walls of different types of cells.

Protocol

1. Stem Embedding

1. Make a 7% agarose solution in water (7 g of electrophoresis-grade agarose in 100 ml distilled water). Dissolve the agarose by autoclaving for 20 min or by microwaving for 20 min at lowest intensity (e.g., 10% intensity of a 1,250-watt microwave).
2. Prepare a homemade mold for embedding the stems using plastic vials.
   1. Using a razor blade, cut off the conical bottom of a 2 ml screw-cap microcentrifuge tube (Part A). With a syringe needle, puncture a hole in the tube cap slightly larger than the diameter of the stem to be embedded. Next, cut 0.5 cm off the bottom of a 0.6 ml microcentrifuge tube (Part B).
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Results

Figure 1. Homemade mold for embedding the stems. On the left side; top of the 2 ml screw-cap microcentrifuge tube (Part A) and the bottom from the 0.6 ml microcentrifuge tube (Part B). On the right side; the Parafilm holding the two parts; A and B to form the final mold.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agarose  	EMD	MERC2125	 CAS Number: 9012-36-6
Phloroglucinol  	Sigma	P 3502	 1,3,5-trihydroxybenzene [CAS Number: 108-73-6]
Hydrochloric acid  	 EMD	HX0603-75	CAS Number: 7647-01-0
Ethanol 190 proof   	KOPTEC	V1401	CAS Number: 64-17-5
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