Ti u camera

The accumulation of ROS was detected in fungal spores protoplast using a 2′,7′-dichlorofluorescin diacetate (DCFH-DA) staining protocol (Chang et al., 2011 (link)). The DCFH-DA, a cell-permeable non-fluorescent probe that turns to highly fluorescent 2′,7′-dichlorofluorescein in the presence of ROS (Sigma-Aldrich, United States) was dissolved into dimethyl sulfoxide (DMSO), and the stock solution was conserved at −20°C. Fungal protoplasts, previously washed with sterile deionized water, were incubated into 1 mL of freshly prepared solution of 10 μM DCFH-DA in phosphate-buffered saline (PBS) for 4 h at 28°C in the dark. Later, the protoplasts were collected by a brief centrifugation and washed twice with PBS and subsequently examined under a Nikon Eclipse Ti-U fluorescent microscope. Micrographs were taken at a magnification of 10 × using a Nikon Ti-U camera. The fluorescence intensity was analyzed using the software Image-Pro Plus 6.0.

Freehand cross-sections were made with a razor blade from healthy and TCDD-exposed roots of date palm seedlings. Sections were fixed overnight in a formaldehyde/acetic acid/alcohol 10/5/85 (v/v) solution at 4 °C. Sections were stained for 1 min in a fresh solution of 0.1% (w/v) neutral red (Sigma-Aldrich) in 0.01 M phosphate buffer (pH 6.5), which specifically dyes suberin in epidermal cell walls. Sections were then examined under a Nikon Eclipse Ti-U fluorescent microscope and micrographs recorded at a magnification of 10× a using a Nikon Ti-U camera.