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

Published: April 30, 2023

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 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). Prepare a homemade mold for embedding the stems using plastic vials. 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…

Representative 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. Figure 2. Phloroglucinol-HCl sta…

Declarações

The authors have nothing to disclose.

Materials

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

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Wiesner Staining for Visualizing Lignin: A Technique to Stain Lignin Deposits Present in Stems of Arabidopsis Thaliana. J. Vis. Exp. (Pending Publication), e21057, doi: (2023).

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