Ds oimc camera

Following treatment, 10-day-old seedlings grown in MS medium in 12-well microtiter plates were fixed in a 3:1 ethanol:acetic acid solution for several hours. The fixative was changed several times to ensure both thorough fixing and clearing of the tissues, which is essential for good callose detection in the leaves. The seedlings were rehydrated in 70% ethanol for 2 h, 50% ethanol for an additional 2 h, and water overnight. After two or three water washes, the seedlings were treated with 10% NaOH and held at 37°C for 1 to 2 h to render the tissues transparent. After three or four water washes, the seedlings were incubated in 150 mM K₂HPO₄ (pH 9.5) and 0.01% aniline blue (Sigma-Aldrich) for several hours. The leaves were mounted on slides with 50% glycerol, and callose was observed immediately using a fluorescence microscope (Nikon Eclipse 80i) under UV light (excitation, 350 nm; emission, 420 nm). Pictures were taken with a Nikon DS-oiMc camera and analyzed by NIS elements BR3.10 software. For callose counting, pictures from three independent biological repeats were used, with callose being counted in four areas of each picture.

Following treatment, 10-day-old seedlings grown in MS medium in 12-well microtiter plates were fixed in a 3:1 ethanol:acetic acid solution for several hours. The fixative was changed several times to ensure both thorough fixing and clearing of the tissues, which is essential for good callose detection in the leaves. The seedlings were rehydrated in 70% ethanol for 2 h, 50% ethanol for an additional 2 h, and water overnight. After two or three water washes, the seedlings were treated with 10% NaOH and held at 37°C for 1 to 2 h to render the tissues transparent. After three or four water washes, the seedlings were incubated in 150 mM K₂HPO₄ (pH 9.5) and 0.01% aniline blue (Sigma-Aldrich) for several hours. The leaves were mounted on slides with 50% glycerol, and callose was observed immediately using a fluorescence microscope (Nikon Eclipse 80i) under UV light (excitation, 350 nm; emission, 420 nm). Pictures were taken with a Nikon DS-oiMc camera and analyzed by NIS elements BR3.10 software. For callose counting, pictures from three independent biological repeats were used, with callose being counted in four areas of each picture.