Axioskop 2 plus system

Floral organs were immersed in 90% acetone, and stored for one or two nights at -20°C. Then, 90% acetone was replaced by 1 phosphate buffer (100 mM NaPO 4 ; 10 stock solution [1M NaPO 4 ] was prepared by mixing 1M Na 2 HPO 4 and 1M NaH 2 PO 4 at a ratio of 61:39; 10 stock solution was diluted 10 times and used as 1 phosphate buffer), which was immediately replaced by the GUS-staining solution (100 mM NaPO 4 [pH 7.4], 10 mM EDTA, 0.1% Triton X-100, 1 mM potassium ferricyanide, 1 mM potassium ferrocyanide, 0.5 mg/mL 5-bromo-4-chloro-3-indolyl-β-D-glucuronide cyclohexylammonium). The GUS-staining solution was vacuum infiltrated into the floral organs for 5 min at 20-25°C. The floral organs infiltrated by the GUS-staining solution were incubated for 16 h in the dark at 37°C for GUS-staining reaction. The GUS-staining solution was replaced by 1 phosphate buffer, followed by rinsing with 70% ethanol two times. After rinsing, an ethanol and acetic acid mixture (ethanol : acetic acid = 6:1) was added for specimen preparation. The specimens were mounted with chloral hydrate solution (chloral hydrate: glycerol: water = 8 [g] : 1 [ml] : 2 [ml]) and examined using a differencial interference contrast microscope (DIC) (Axioskop 2 plus system [Carl Zeiss] and a high-sensitivity cooled CCD color camera VB-7010
[Keyence]).