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In This Article

  • Overview
  • Protocol
  • Results
  • Disclosures
  • Materials
  • References

Overview

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

  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 (....

Results

figure-results-25
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.

....

Disclosures

No conflicts of interest declared.

Materials

NameCompanyCatalog NumberComments
AgaroseEMDMERC2125CAS Number: 9012-36-6
Fluorescent Brightener 28/ Calcofluor White StainSigmaF35434,4'-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]
VibratomeLeicaLeica Vibrating blade microtome VT1000Shttp://www.leicabiosystems.com/products/sectioning/vibrating-blade-microtomes/details/product/leica-vt1000-s/
RazorAmerican Safety razor companyItem # 60-0139-0000Stainless Steel Double Edge Blade (Personna Super)
Screw Cap Microcentrifuge Tubes (2 ml)VWR16466-044
Microcentrifuge Tubes (0.6 ml)Axygen ScientificMCT-060-C
MittBel-Art380000000SCIENCEWARE Hot Hand Protector Mitt
Tissue adhesiveTed Pella Inc10033Store at 4 °C or 20 °C for 3 months or longer storage
MicrowavePanasonicNN-SD762SPELCO Pro CA 44 Instant tissue adhesive
Micro Cover Glasses, Square, No. 1VWR48366-06722 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 mmVWR48312-00375 x 25 mm - 1 mm
Parafilm MAlcan packagingBRNDPM998

References

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