Congo Red Staining: A Technique to Visualize Cellulose Deposits in Plant Stem Sections

Published: April 30, 2023

Abstract

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

In this video, we demonstrate the Congo red staining method to visualize cellulose deposits in Arabidopsis thaliana stem sections.

Protocol

1. Congo Red Staining

  1. Dissolve 0.5 g congo red in 100 ml of distilled water to prepare a 0.5% congo red solution.
  2. Transfer stem sections to a 2.0 ml microcentrifuge tube. Add 1 ml of the 0.5% congo red solution to the tube. Gently pipette the 0.5% congo red solution up and down without disturbing the sections.
    NOTE: Be careful not to pipette the sections into the pipette tip as this may damage the sections.
  3. Incubate at room temperature for 10 min and repeat the pipetting. Follow with another 3 min of incubation at room temperature.
  4. Use a 1 ml pipette to draw out 700 µl of 0.5% congo red solution. Add 700 µl of distilled water to rinse out the congo red solution. Repeat the wash 3-4x until the washing solution is clear.
  5. Use a 1 ml pipette with its tip cut in such a way that the sections can be pipetted out easily without damage. Gently pipette sections into the tip and onto a microscope slide and cover them with coverslip. Observe the samples on the microscope slide under blue-light excitation using a filter with a bandpass of 560/40.
    NOTE: Do not store the sections in water for long prior to the microscopic analysis, as the water will wash out the congo red in 10-30 min.

Disclosures

The authors have nothing to disclose.

Materials

Congo Red Sigma  C6277 Disodium 3,3'-[[1,1'-biphenyl]-4,4'-diylbis(azo)]bis(4-aminonaphthalene 1-sulphonate) [CAS Number 573-58-0]
Microcentrifuge Tubes (0.6 ml) Axygen Scientific MCT-060-C
Camera with CCD chip with no mechanical shutter  Hamamatsu C4742-95
High speed color camera    QImaging MicroPublisher 5.0 RTV
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 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
TX2 Filter cube Leica 11513851/11513885 Filter used for Congo red analysis with a band-pass of 560/40.

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Cite This Article
Congo Red Staining: A Technique to Visualize Cellulose Deposits in Plant Stem Sections. J. Vis. Exp. (Pending Publication), e21059, doi: (2023).

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