Pollen Germination Analysis via Aniline Blue

Aniline blue staining was carried out to explore pollen germination according to previous descriptions [60 (link)], with slight modifications. After 24 h of hand pollination, pistils were fixed overnight in Carnoy’s fixative solution. Fixed pistils were washed with PBS buffer three times for 20 min each time and treated with 5 M NaOH solution for 24 h. Then, the samples were rewashed with PBS buffer three times and stained with 0.1% aniline blue for 4 h under dark conditions. The stained pistils were observed using a TCS SP8 confocal laser microscope (Leica) for fluorescence observation. The fluorescence was excited using a 405 nm laser.