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

Materials

Name	Company	Catalog Number	Comments
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.