The zona pellucida of embryos was removed using acid Tyrode solution (Sigma-Aldrich) and then fixed and permeabilized with 2% PFA-PVA containing 0.25% Triton X-100 for 10 min on ice. The samples were placed on glass slides, evaporated to dryness, dehydrated sequentially in 70 and 100% ethanol and then air-dried. Hybridization buffer containing an Xist probe (provided by T. Sado) was prepared using a Nick Translation Kit (Abbott, Abbott Park, IL, USA) and Cy3-dUTP (GE Healthcare Life Sciences, Fairfield, CT, USA) and was then applied to the slides. The slides were then incubated and washed as previously described26 (link). Fluorescence was visualized using the LSM510.
Acid tyrode solution
Manufactured by Merck Group
Acid Tyrode solution is a buffered salt solution commonly used in physiological research. It serves as a balanced salt solution to maintain the appropriate pH and osmolarity for cell and tissue cultures.
Automatically generated - may contain errors
Lab products found in correlation
Apotome, by Zeiss (2 mentions)
Cy3 dutp, by GE Healthcare (1 mentions)
Nick translation kit, by Abbott (1 mentions)
Tryple solution, by Thermo Fisher Scientific (1 mentions)
Esg1107, by Merck Group (1 mentions)
Rnaseout, by Thermo Fisher Scientific (1 mentions)
Anti cdx2, by BioGenex (1 mentions)
Alexa fluor 594 phalloidin, by Thermo Fisher Scientific (1 mentions)
Anti oct3 4 antibody, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole dapi, by Agilent Technologies (1 mentions)
Axiovision software 4, by Zeiss (1 mentions)
Axiovert 200m fluorescence microscope, by Zeiss (1 mentions)
710 meta, by Zeiss (1 mentions)
M2 medium, by Merck Group (1 mentions)
5 protocols using acid tyrode solution
1
Xist RNA Localization in Embryos
2
Derivation and Culture of Mouse ESCs
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Mouse ESCs were derived as previously reported (40 (link)). Briefly, embryonic day 3.5 blastocysts were recovered from Lnc956+/− females mated with Lnc956−/− male mice. After removal of zona pellucida with acid tyrode solution (100 ml; Sigma-Aldrich, T1788), blastocysts were plated on mitomycin C–treated mouse embryonic fibroblasts (MEFs) and cultured for 5 days. The outgrowths were then picked with a mouth pipette and transferred into 50-μl droplet of 0.05% TrypLE solution (Gibco, 12604021) for 3 min. Dissociated cells were pipetted into 48-well culture plates with a MEF feeder for further culture. After routine clonal expansion, genotype was confirmed by PCR with specific primers (table S1).
Mouse ESCs were maintained in Dulbecco’s modified Eagle's medium (DMEM)/F12 supplemented with 20% KO serum replacement (Thermo Fisher Scientific, A3181502), recombinant mouse leukemia inhibitory factor (104 U/ml; Millipore, ESG1107), 2 mMl -glutamine (Sigma-Aldrich, G8540-25g), penicillin (100 U/ml)/streptomycin (100 μg/ml) (Gibco, 15140-122), 100 μM β-mercaptoethanol (Sigma-Aldrich, M7522), and 1× MEM nonessential amino acids solution (Gibco, 11140-035).
Mouse ESCs were maintained in Dulbecco’s modified Eagle's medium (DMEM)/F12 supplemented with 20% KO serum replacement (Thermo Fisher Scientific, A3181502), recombinant mouse leukemia inhibitory factor (104 U/ml; Millipore, ESG1107), 2 mM
3
Immunofluorescent Analysis of Preimplantation Embryos
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The zona pellucida of embryos was removed using acid Tyrode solution (Sigma-Aldrich) and fixed with 2% PFA-PVA for 15 min at RT in four-well dishes. The fixed samples were permeabilized with 0.5% Triton X-100 in PBS-PVA for 20 min on ice. After washing with PBS-PVA, the samples were blocked in 1% BSA-PBS-PVA containing 1.3 U ml−1 RNaseOUT (Life Technologies) for 30 min at RT. After washing, the embryos were incubated with primary antibodies (anti-CDX2 (BioGenex, San Ramon, CA, USA), diluted 1:30 and anti-H3K27me3 diluted 1:150 in blocking buffer containing 1.3 U ml−1 RNaseOUT) for 1 h at RT. Secondary antibody reactions were performed as described above. The samples were placed on glass slides, evaporated to dryness, dehydrated sequentially in 70 and 100% ethanol and air dried. The samples were then analysed by FISH according to the procedures described above.
4
Blastocyst Cell Counting Using Immunofluorescence
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The number of cells in each blastocyst in all groups was counted by first staining with anti-Oct3/4 antibody (Santa Cruz) to identify inner cell mass (ICM) cells and counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Dako, USA) to label nuclei. Five days after IVF, the zona pellucida (ZP) was removed using an acid Tyrode solution (pH 2.5; Sigma-Aldrich). ZP-free blastocysts were fixed in 3.7% paraformaldehyde in 0.02% Triton X-100 in DPBS containing 0.1% BSA (DPBS-BSA) for 30 min at 4°C and permeabilized with 0.1% Triton X-100. Blastocysts were transferred to 5% normal goat serum for 2–4 h at 4°C and then incubated overnight at 4°C with anti-Oct3/4 antibody (Santa Cruz). Blastocysts were then incubated with secondary antibody and co-stained with Alexa Fluor 594 Phalloidin (Molecular Probes, USA) and DAPI. Blastocysts were mounted on a glass-bottomed dish in PBS and imaged using a Zeiss Axiovert 200M fluorescence microscope with Apotome and a 40× oil-immersion objective lens (Carl Zeiss, Germany). Z-stack images (15–20) of individual blastocysts were obtained and analyzed using Axiovision software 4.6 (Carl Zeiss). Oct3/4- and DAPI-positive cells were counted as ICMs; DAPI-only positive cells were counted as the total cell number. The number of trophectoderms (TEs) was calculated as total cell number minus ICM number.
5
Immunofluorescence Localization of Bub3 in Mouse Oocytes
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Chromosome spread was performed as described previously [26] . Briefly, the zona pellucida of MII eggs was removed by acid Tyrode solution (Sigma). After washing two to three times in M2 medium (Sigma), the oocytes were transferred to glass slides dipped in a solution of 1% paraformaldehyde in distilled H 2 O (pH 9.2) containing 0.15% Triton X-100 and 3 mM dithiothreitol. After the slides were dry, they were blocked with 1% BSA for 1 h at room temperature and incubated in anti-Bub3 antibody overnight at 48C, washed three times and incubated with FITC-conjugated goat anti-rabbit IgG antibody (1:100) for 2 h at room temperature. DNA was stained with PI for 10 min. A laser scanning confocal microscope (Zeiss 710 META) was used for microscopy and image analysis.
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