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.
Divulgazioni
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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Citazione di questo articolo
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).