Double-Staining Method: A Technique to Visualize Plant-Fungus Interactions Using Ruthenium Red and Lactophenol Cotton Blue

Abstract

Source: Marques, J. P. R., and Nuevo, L. G. Double-Staining Method to Detect Pectin in Plant-Fungus Interaction. J. Vis. Exp. (2022).

This video demonstrates a double staining method using ruthenium red and lactophenol cotton blue to visualize pectins and chitins during plant-fungus interactions in a coffee rust plant. The procedure helps understand plant defense mechanisms during fungal infections.

Protocol

1. Double staining process

1. Begin with ultrathin, resin-embedded leaf sections of Coffee arabica cv. Catuaí infected with coffee rust. Cover the sections with 2 mL of 5% cotton blue in lactophenol (40% glycerol, 20% phenol, and 20% lactic acid in water) and heat them on a hot plate at 45 °C for 5 min (Figure 1A).
2. Remove the excess dye by washing the slide three times in a beaker filled with distilled water (Figure 1B-D).
3. Stain with 2 mL of 0.01% ruthenium red in water for 1 min (Figure 1E).
4. Remove the excess dye by washing the slide three times in a beaker filled with distilled water (Figure 1F,G).
5. Put a drop of distilled water over the sections and cover the sections with a 24 mm x 60 mm coverslip for performing light microscopy analysis.

Results

Figure 1: Details of the individual steps of the double-staining protocol. (A) Cover the sections with drops of cotton blue lactophenol and heat the slides on a hot plate at 45 °C. (B-C) Remove the excess dye by washing in distilled water. (D

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Hot plate	Dubesser	SSCD25X30-110V	Staining
Cotton Blue Lactophenol	Metaquímica	70SOLSIG024629	Staining
Microscopy	Zeiss	#490040-0030-000	Image capture
Plastic molding cup tray	EMS	10176-30	Staining
Ruthenium red	LABHouse	#006004	Staining
Software Axion Vision	Zeiss	#410130-0909-000	Image capture