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

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.

النتائج

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) Sections on the glass slides after washing (denoted by arrows). (E) Cover the sections with drops of ruthenium...

Materials

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