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

Cut the block into 5-micrometer thick sections using a rotative microtome equipped with 8-centimeter steel blades. Place the sections onto glass slides covered with distilled water, then, transfer the slides to a hot plate to dry at 40 degrees Celsius, and promote the adhesion of the sections to the glass slides.

After drying, label the glass slides with the block reference name and the slide number. Cover the sections with 2 milliliters of 5% cotton blue in lactophenol, and heat them on a hot plate at 45 degrees Celsius for five minutes.

Remove the excess dye by washing the slide three times in a beaker filled with distilled water. Stain it with 2 milliliters of 0.01% ruthenium red in water for one minute.

Remove the excess dye by washing the slide three times in a beaker filled with distilled water. Put a drop of distilled water over the sections, and cover the sections with a coverslip for performing light microscopy analysis.