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
- 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).
- Remove the excess dye by washing the slide three times in a beaker filled with distilled water (Figure 1B-D).
- Stain with 2 mL of 0.01% ruthenium red in water for 1 min (Figure 1E).
- Remove the excess dye by washing the slide three times in a beaker filled with distilled water (Figure 1F,G).
- 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.
Representative 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) Sections on the glass slides after washing (denoted by arrows). (E) Cover the sections with drops of ruthenium red. (F-G) Remove the excess dye by washing in distilled water.
Divulgaciones
The authors have nothing to disclose.
Materials
| 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 |
