Lsm 5 pascal confocal laser scanning microscopy

The reporters were used for analyzing the expression of E2F genes. The construct of pE2F:E2F-VENUS was a translational fusion of E2F to VENUS which was driven by its native promoter (∼2 kb). NOS terminator and VENUS were amplified by PCR and consecutively inserted into the PstI-HindIII site and the SalI-PstI site of pCAMBIA1300 to generate pCAMBIA1300-VENUS. Subsequently, the genomic DNA sequence of a E2F gene was amplified by PCR and integrated into the SalI site of pCAMBIA1300-VENUS using the pEASY Uni-Seamless Cloning and Assembly Kit (TransGen Biotech, Beijing, China) to generate pE2F:E2F-VENUS. The primers used for construction of pE2F:E2F-VENUS are listed in Supplementary Table S1. To visualize the expression pattern of a reporter, the fluorescence was excited at 488 nm and collected with a 515∼530 nm bandpass filter using a Zeiss LSM 5 Pascal Confocal Laser Scanning Microscopy (Germany).

The procedure used to analyze female gametophyte development was carried out as described (Christensen et al., 1997 (link)). Briefly, pistils were fixed in the fixative solution (4% glutaraldehyde and 12.5 mM cacodylic acid, pH 6.9) for 4 h. The tissues were dehydrated in a series of increasing concentrations of ethanol (10, 20, 40, 60, 80, and 95%, each for 10 min) and kept in 95% ethanol overnight. The tissues were then washed with 100% ethanol twice, each for 10 min. After dehydration, the tissues were cleared in the benzyl benzoate/benzyl alcohol (2:1) solution for 20 min. Ovules were dissected, mounted in immersion oil, and observed at the excitation wavelength 488 nm and the emission wavelength 515∼530 nm using a Carl Zeiss LSM 5 Pascal Confocal Laser Scanning Microscopy (Germany).