Rabbit anti trp53

Immunofluorescence was performed as previously described (Cui et al. 2016a (link), Cui et al. 2016b (link)). In vivo derived blastocysts were flushed at E3.5, and then cultured overnight before fixation and immunofluorescence (to ensure embryos had undergone EPI/PE/TE specification). In vitro blastocysts were harvested at 4 days post microinjection. Primary antibodies used in this study include: mouse anti-Cdx2 (BioGenex, MU392A-UC); rabbit anti-Nanog (abcam, ab80892); rabbit anti-Trp53 (Cell Signaling Technology, #9284); goat anti-Sox17 (R&D Systems, AF1924); goat anti-Oct4 (abcam, ab27985). After secondary antibodies (Alexa Fluor, Life Technologies) and DAPI (Sigma) staining, embryos were transferred to chambered slides (BD Falcon) with 1 embryo per well for imaging. Embryos were imaged using Nikon A1 Spectral Detector Confocal with FLIM Module. Z-stacks (20X objective, 8 μm sections) were collected and maximum projection was applied. Blastocysts collected from heterozygous intercrosses were imaged prior to knowledge of their genotypes. After imaging, embryos were individually recovered and lysed for genotyping.

Immunofluorescence staining (IF) was carried out in accordance with the methods of our previous studies (Miao et al. 2020 (link)). E3.0 morula embryos were harvested and then fixed directly in 4% paraformaldehyde. After brief wash in PBS, embryos were permeabilized with 0.5% Triton X-100 for 20 min. Embryos were then blocked in blocking solution (PBS with 10% FBS and 0.1% Triton) for 1 h at room temperature, and then incubated with primary antibodies overnight at 4 °C. All primary antibodies were diluted 1:200 using the blocking solution, including: goat anti-OCT4 (abcam, ab27985); mouse anti-CDX2 (BioGenex, MU392A-UC); rabbit anti-TRP53 (Cell Signaling Technology, #9284). After 3 washes, embryos were incubated with suitable secondary antibodies (Alexa Fluor, Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature in dark. After 2 washes, DNA was stained with 4,6-diamidino-2-phenylindole (DAPI) before transferring embryos into single wells of chambered slides (Corning Co., Corning, NY, USA) for imaging under a Nikon A1 Spectral Detector Confocal with FLIM Module. Z-stacks (20X objective, 8 μm sections) were collected and maximum projection applied. Embryos were handled individually such that each one was imaged and then recovered for PCR genotyping.