Ruthenium red is a polycationic stain used to visualize acid polysaccharides on the outer surface of cells and and elastic tissue fibers.

Store at 4°C.

Protocol for seed coat Ruthenium Red staining

1. Prepare Ruthenium Red work solution

0.01% ruthenium red

0.1 g in 1 L dH2O (pH 6.5 -7.5)

Stored at 4 ºC

Discard solution if no longer bright pink (It may turn brownish, orange, purple.)

2. Place ~20 seeds in a clean microfuge tube. Seeds should be completely desiccated (1-2 weeks after harvesting minimum) before staining.

3. Add 800 μl dH₂O or 50 mM EDTA.

4. Shake vigorously (~400 rpm) on an orbital shaker for 2 h at room temperature to hydrate, and therefore release the mucilage from the epidermal seed coat

5. Remove as much of the first solution as possible with a pipet or an aspirator and replace with 800 μl of 0.01% ruthenium red solution.

6. Shake vigorously (~400 rpm) on an orbital shaker for 1 h at room temperature.

7. Remove the ruthenium red solution and replace with dH₂O.

8. Observe the seeds, mounted in water, under a dissecting microscope in a white porcelin depression plate, or on a clean depression s

Ruthenium red is a polycationic stain used to visualize acid polysaccharides on the outer surface of cells and and elastic tissue fibers.

[1] Waller LN, Fox N, Fox KF, Fox A, Price RL. Ruthenium red staining for ultrastructural visualization of a glycoprotein layer surrounding the spore of Bacillus anthracis and Bacillus subtilis. J Microbiol Methods. 2004 Jul;58(1):23-30. doi: 10.1016/j.mimet.2004.02.012. PMID: 15177900.