Double-Staining Method: A Technique to Visualize Plant-Fungus Interactions Using Ruthenium Red and Lactophenol Cotton Blue
Published: April 30, 2023
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).<…
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…
Offenlegungen
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 |
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Double-Staining Method: A Technique to Visualize Plant-Fungus Interactions Using Ruthenium Red and Lactophenol Cotton Blue. J. Vis. Exp. (Pending Publication), e21054, doi: (2023).