2.4 Anther staining and confocal microscopy
The spikelets were dissected from the spike, and anthers were removed
from each spikelet under a stereo microscope (S6D, Leica, Germany).
Depending on the experiment situation, anthers dipped in absolute
ethanol for 30 min. Application of absolute ethanol was repeated thrice
after 30 min until tissue change into white color. Hyphae in anthers
were stained with chitin specific dye WGA-AF 488 (Invitrogen, Eugene,
USA) and anther cells were stained with propidium iodide (PI)
(Invitrogen, Eugene, USA). Samples were incubated at 25oC for 60 min in 0.02% Tween 20 containing WGA-AF488
and PI at the ratio of 1:2. After staining, samples were rinsed 4 to 6
times in 1× PBS (pH 7.4) (Suolaibao, Beijing, China) and stored in PBS
at 4 °C without light. The fungal staining was performed at 25 °C.
Samples were observed under a confocal laser scanning microscope (Leica
SP8, Germany) with excitation 488 nm/emission 510-550 nm (for WGA-AF
488) and excitation 561 nm/emission 570-730 nm (for PI).