Optimized Expansion Microscopy of Arabidopsis Seeds

The expansion microscopy technique [109 ] optimized for Arabidopsis seeds was conducted as previously described [73 (link)]. Anti-Fibrillarin antibody (ab4566, Abcam) and anti-alpha Tubulin antibody (ab89984, Abcam) were used in 1:500 dilution as primary antibodies. Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150113, Abcam) and goat Anti-Chicken IgY H&L (Alexa Fluor® 555) (ab150170, Abcam) were used in 1:500 dilution as secondary antibodies. For each sample, a stack of nine images with 1-μm intervals were recorded by ZEISS LSM700 with 25× oil objective and ZEN software at 1024 × 1024 resolution in 8-bit. DAPI signals were excited by 405-nm laser and passed through SP490 filters. Alexa488 signals were excited by 488-nm laser and passed through BP490-635 filters. Alexa555 signals were excited by 555-nm laser and passed through 560–1000-nm filters. Pinhole sizes were kept as 1 airy unit for each color, and color channels were scanned separately. FIJI software was used for image processing and nuclear size quantification. Each stack of images was first Z-projected on maximum intensity and then the nuclear areas were determined based on DAPI signals. The zygotic nuclei were distinguished from the endosperm nuclei according to position and tubulin staining patterns.