Immunofluorescence images

In order to examine histone modifications at very early stages, such as 0 mpf, 15 mpf and 30 mpf (minutes post fertilization), embryos were obtained via in vitro fertilization. Then embryos at the exact time point after fertilization were fixed by 4% polyformaldehyde overnight at 4℃. For immunofluorescence of α-amanitin injected embryos, embryos naturally fertilized were microinjected with α-amanitin and fixed at desired stages. Then they were dechorionated manually and dehydrated with methanol. The whole-mount immunofluorescences with H3K4me3 antibody (Homemade), H3K27ac antibody (Active Motif, Cat # 39133), H3K27me3 antibody (Active Motif, Cat # 61017), H3K36me3 antibody (Active Motif, Cat # 61021), Dnmt1 antibody (Santa Cruz, Cat # sc-20701), and DNA 5mC antibody (Abcam, Cat # ab10805) were done with DAPI (Invitrogen, Cat # D1306) staining and performed essentially as before (Wu et al., 2014). The secondary antibodies were Alexa Fluor® 488 conjugated anti-rabbit and Alexa Fluor® 488 conjugated anti-mouse (Jackson ImmunoResearch, 1:200 diluted). After staining, embryos were deyolked by tweezers and mounted on glass slides in mounting medium (Sigma, Cat # P3130) at animal polar upturned position. Images were acquired on 710 META laser scanning confocal microscope and manipulated by ZEN software.
Treated or untreated embryos were anesthetized at desired stages with 0.02% tricaine and mounted in 5% methyl cellulose (Sigma, Cat # M-6385) for observation and phenotype pictures were taken under Nikon SMZ1500 microscope.