Arabidopsis Crossing and Aniline Blue Staining

Stage 12 flowers were emasculated, and stigmas were pollinated with desired pollen grains (Smyth et al., 1990). For tracking pollen tube growth into the stigma, aniline blue staining was conducted 8 h after pollination. Pollinated pistils were fixed with 10% acetic acid in ethanol for 12 h. The samples were washed three times with distilled water, and, subsequently, pistils were incubated in 5 m NaOH overnight. Pistils were then washed three times with distilled water and stained for ∼15 h with aniline blue (Fisher Scientific) [0.1% (w/v) aniline blue in 0.1 m sodium phosphate buffer, pH 7.5] in the dark. After staining, pistils are placed in a drop of 50% glycerin on a microscope slide and covered with a coverslip. Fluorescence of pollen tubes was detected using a confocal microscope (excitation: 405 nm; emission: 440 to 550 nm).