The procedures of probe making and the FISH experiment were described previously (Inoue et al. 2017b (link)). Morula embryos were obtained by IVF with BDF1 sperm and fixed at 78 hpf. E3.5, E4.0, and E6.5 embryos were obtained by natural mating with BDF1 males. The day of plug was counted as E0.5. E3.5 and E4.0 embryos were flushed out from the uterus at noon of the third day, cultured in KSOM (Millipore), and fixed at noon (E3.5) or midnight (E4.0). For E6.5 embryos, ExE was dissected as described previously (Sugimoto et al. 2015 (link)). For some experiments, embryos were immunostained with rabbit anti-Nanog antibody (1/100; Cosmobio, RCAB002pF) or rabbit anti-H3K27me3 antibody (1/500; Millipore, 07449) after completion of the Xist RNA FISH procedure. The secondary antibodies were Alexa flour 488 donkey anti-rabbit IgG (Life Technologies) and Alexa flour 647 donkey anti-rabbit IgG (Life Technologies).
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Xist RNA FISH and Immunostaining in Mouse Embryos
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Other organizations : Howard Hughes Medical Institute, Boston Children's Hospital, Harvard Stem Cell Institute, Harvard University
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Variable analysis
independent variables
- Methods of obtaining embryos (IVF, natural mating)
- Developmental stages of embryos (morula, E3.5, E4.0, E6.5)
- Expression of Xist RNA (measured by FISH)
- Expression of Nanog (measured by immunostaining)
- Expression of H3K27me3 (measured by immunostaining)
- Strains of mice (BDF1)
- Fixation time points of embryos
- Culture conditions (KSOM)
- Not explicitly mentioned
- Not explicitly mentioned
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