Calcofluor White Staining: A Simple Method for UV-Induced Fluorescence Visualization of Cellulose Deposits in Cell Walls of Dicot Plant Stem Sections

Published: April 30, 2023

Abstract

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

In this video, we describe a staining procedure to visualize cellulose deposition within cell walls of Arabidopsis thaliana, a dicot plant, using calcofluor white, which specifically binds to β-linked glucans, exhibiting fluorescence under UV light.

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…

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.

Divulgaciones

The authors have nothing to disclose.

Materials

Agarose EMD MERC2125 CAS Number: 9012-36-6
Fluorescent Brightener 28/ Calcofluor White Stain Sigma F3543 4,4'-Bis[[4-[bis(2-hydroxyethyl)amino]-6-anilino-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonic acid [CAS Number 4404-43-7]
Vibratome Leica Leica Vibrating blade microtome VT1000S http://www.leicabiosystems.com/products/sectioning/vibrating-blade-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
Mitt Bel-Art 380000000 SCIENCEWARE Hot Hand Protector Mitt
Tissue adhesive Ted Pella Inc 10033 Store at 4 °C or 20 °C for 3 months or longer storage
Microwave Panasonic NN-SD762S PELCO Pro CA 44 Instant tissue adhesive
Micro Cover Glasses, Square, No. 1 VWR 48366-067 22 x 22 mm (7/8 x 7/8")-Cover glasses are corrosion-resistant and uniformly thick and flat. No. 1 thickness is 0.13 to 0.17 mm.
Frosted Micro Slides, 1 mm VWR 48312-003 75 x 25 mm – 1 mm
Parafilm M Alcan packaging BRNDPM998

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Calcofluor White Staining: A Simple Method for UV-Induced Fluorescence Visualization of Cellulose Deposits in Cell Walls of Dicot Plant Stem Sections. J. Vis. Exp. (Pending Publication), e21060, doi: (2023).

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