The processes were performed as described previously [19 (link)]. MII-stage oocytes matured in vivo were collected 14 hours later from the Fallopian tubes, and digested with 1 mg/ml hyaluronidase (Sigma, USA). The zona pellucida of MII-stage oocytes matured in vivo were removed with acid Tyrode’s solution (Sigma, USA). The oocytes without zona pellucida were fixed in 1% paraformaldehyde with 0.15% Triton X-100 and 3 mM dithiothreitol on glass slides. The slides were dried at room temperature and blocked with 1% BSA for 1 h, then incubated in anticentromere antibodies, (ACA; 1:50) at 4° C overnight. After washing, the slides were incubated with secondary antibodies Cy5 (1:200) for 1 h at room temperature. The nuclei were stained with DAPI at room temperature for 15 min. DABICO was added and coverslips were used for lightly covering the samples.
Acid tyrode s solution
Manufactured by Merck Group
Sourced in United States
Acid Tyrode's solution is a widely used buffer solution in cell and tissue culture laboratories. It is a balanced salt solution that mimics the ionic composition of human extracellular fluid. The solution is designed to maintain the appropriate pH and osmotic pressure for the cultivation of cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
M2 medium, by Merck Group (5 mentions)
Mineral oil, by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Hyaluronidase, by Merck Group (2 mentions)
Dissecting microscope, by Leica (2 mentions)
Anti mouse rabbit serum, by Merck Group (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Chick serum, by Merck Group (2 mentions)
Heparin, by Merck Group (2 mentions)
Hoechst 33342, by Merck Group (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Female cd 1 mice, by Charles River Laboratories (2 mentions)
Ksom medium, by Merck Group (2 mentions)
Cd1 male mice, by Charles River Laboratories (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Hoechst, by Merck Group (1 mentions)
Anti oct4, by Santa Cruz Biotechnology (1 mentions)
Anti cdx2, by BioGenex (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
56 protocols using acid tyrode s solution
1
Oocyte Fixation and Immunostaining
2
Immunostaining of Mouse Embryos
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Mouse embryos were rinsed in Acid Tyrode’s solution, (Sigma Aldrich) and fixed in 3.7% formaldehyde on ice for 30 minutes. Primary antibodies used were anti-Oct4 (1:250, Santa Cruz Biotechnology) and anti-Cdx2 (1:200, BioGenex) and secondaries were Alexa555 and CruzFlur647 (see Table D in S1 Text ). DNA was stained with Hoechst (2μg/ml, Sigma Aldrich). Embryos were placed on a glass slide coated with a 1% agarose pad and compressed to a 3:1 aspect ratio. All confocal images were acquired using a 63x 1.4 NA objective, on an Axioobserver Z1 Zeiss 780 confocal microscope with Zen2009 software. Z-stack images were acquired at 0.3μm intervals.
3
Generating Chimeric Mice via Embryo Aggregation
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Chimeras were generated by diploid embryo aggregation. Briefly, E2.5 embryos were collected from superovulating Swiss female mice and the zona pellucida of embryos was removed by treatment with acid Tyrode's solution (Sigma). mESC colonies from control and p120ctn-null mESCs were briefly treated with 0.25% trypsin-EDTA (Invitrogen) to form loosely connected clumps of 7 to 10 cells. Each zona-pellucida free embryo was aggregated with a clump of 7 to 10 cells using depression wells made with an aggregation needle (BLS Ltd, Hungary) in a 35-mm plastic dish (VWR International). Aggregates were cultured overnight in microdrops of KSOM with amino acids (Biognost) under mineral oil (Sigma) at 37°C in 95% air and 5% CO2. The next day, blastocysts were transferred into the uteri of 2.5-dpc pseudopregnant Swiss females previously mated with vasectomized males. Chimeras were identified at birth by the presence of black eyes and later by agouti coat pigmentation.
4
Immunosurgery-based Isolation of Epiblast
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Embryo and cell manipulations were carried out under a dissecting microscope (Leica Microsystems). The zona pellucida was removed using acid Tyrode’s solution (Sigma) at E4.0. E4.5 blastocysts were subjected to immunosurgery as previously described (Solter and Knowles, 1975 (link)). In brief, blastocysts were incubated for 45-60 minutes in a 1:5 dilution of anti-mouse rabbit serum (Sigma) in N2B27, washed in N2B27 and further incubated for 30-60 minutes in a 1:5 dilution of rat serum (in-house) in N2B27 for the complement reaction. The ICM was subsequently cleaned from residual trophectoderm with a narrowly fitting glass pipette. Isolated ICMs were culture in N2B27 at 37°C and 5% CO2 with or without 1 μg/ml Dox an equivalent to E5.5 in which PrE lineage cells surround the matured EPI.
5
Derivation and Propagation of hESC Line
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The zona pellucida of blastocysts was removed by incubation with 0.1% Acid Tyrode’s solution (Sigma-Aldrich)
followed by five washes in hESC culture medium and plating of whole blastocysts on a feeder layer. The
formation of a dome-like shape indicating an inner cell mass (ICM) was monitored for daily. The outgrowth from
the ICM was mechanically removed from the trophectoderm (TE) with a needle at 4 to 5 days after initial
plating and then plated on a new feeder layer. hESC-like cells were propagated by mechanical splitting with a
needle or fine-drawn glass pipette. For propagation, colonies of hESCs were mechanically cut with a 23G needle
into small pieces every 5–7 days, detached from the culture dish and plated onto new feeders, and the culture
medium was changed on a daily basis.
A diploid hESC line (Chula2.hES) derived from frozen-thawed embryos [19 (link)], was cultured and propagated as described above.
The hESC culture medium consisted of knockout DMEM supplemented with 20% Knockout serum replacement (KSR), 1%
GlutaMax, 1% non-essential amino acids, 1% penicillin-streptomycin, and 0.1 mM mercaptoethanol (all from Life
Technologies) and 8 ng/ml bFGF (R&D Systems, Minneapolis, MN, USA).
followed by five washes in hESC culture medium and plating of whole blastocysts on a feeder layer. The
formation of a dome-like shape indicating an inner cell mass (ICM) was monitored for daily. The outgrowth from
the ICM was mechanically removed from the trophectoderm (TE) with a needle at 4 to 5 days after initial
plating and then plated on a new feeder layer. hESC-like cells were propagated by mechanical splitting with a
needle or fine-drawn glass pipette. For propagation, colonies of hESCs were mechanically cut with a 23G needle
into small pieces every 5–7 days, detached from the culture dish and plated onto new feeders, and the culture
medium was changed on a daily basis.
A diploid hESC line (Chula2.hES) derived from frozen-thawed embryos [19 (link)], was cultured and propagated as described above.
The hESC culture medium consisted of knockout DMEM supplemented with 20% Knockout serum replacement (KSR), 1%
GlutaMax, 1% non-essential amino acids, 1% penicillin-streptomycin, and 0.1 mM mercaptoethanol (all from Life
Technologies) and 8 ng/ml bFGF (R&D Systems, Minneapolis, MN, USA).
6
Generation of Chimeric Mice with Wapl Mutation
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ES cell clones containing the target Wapl gene were aggregated for the
generation of chimeric mice. The cell clones were placed on a MEF feeder layer 2 days
before embryo manipulation. The propagated ES cells were dissociated with 0.05% trypsin
just before the aggregation. Eight-cell-stage-embryos were collected at 2.5 dpc from
female ICR mice that had been superovulated by intraperitoneal (i.p.) injection of 5.0 IU
of equine chorionic gonadotropin (Serotropin; ASKA Pharmaceutical, Tokyo, Japan) at 1600
h, followed by injection of 5.0 IU of human chorionic gonadotropin (Gonadotropin 3000;
ASKA Pharmaceutical) 48 h later and then mated naturally with male ICR mice. The zona
pellucida was removed by treatment with acid Tyrode’s solution (Sigma-Aldrich, St. Louis,
MO). Fifteen to twenty ES cells were aggregated with each zona-free embryo and cultured
overnight in micro-drops of KSOM medium (ARK Resource, Kumamoto, Japan) under mineral oil
at 37°C, 5% CO2, and 95% humidity. The chimeric embryos were transferred at 2.5
dpc into the uterine horns of pseudopregnant female ICR mice (Sankyo Labo Service
Corporation). Chimeric mice were identified 20 days after birth by their black eyes and
their black coat color. Chimeric males showing germline transmission were crossed with
C57BL/6 females.
generation of chimeric mice. The cell clones were placed on a MEF feeder layer 2 days
before embryo manipulation. The propagated ES cells were dissociated with 0.05% trypsin
just before the aggregation. Eight-cell-stage-embryos were collected at 2.5 dpc from
female ICR mice that had been superovulated by intraperitoneal (i.p.) injection of 5.0 IU
of equine chorionic gonadotropin (Serotropin; ASKA Pharmaceutical, Tokyo, Japan) at 1600
h, followed by injection of 5.0 IU of human chorionic gonadotropin (Gonadotropin 3000;
ASKA Pharmaceutical) 48 h later and then mated naturally with male ICR mice. The zona
pellucida was removed by treatment with acid Tyrode’s solution (Sigma-Aldrich, St. Louis,
MO). Fifteen to twenty ES cells were aggregated with each zona-free embryo and cultured
overnight in micro-drops of KSOM medium (ARK Resource, Kumamoto, Japan) under mineral oil
at 37°C, 5% CO2, and 95% humidity. The chimeric embryos were transferred at 2.5
dpc into the uterine horns of pseudopregnant female ICR mice (Sankyo Labo Service
Corporation). Chimeric mice were identified 20 days after birth by their black eyes and
their black coat color. Chimeric males showing germline transmission were crossed with
C57BL/6 females.
7
Embryo Aggregate Formation and MEK Inhibition
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Embryos were obtained at 8-cell morula stage by washing E2.5 oviducts with M2 medium (Sigma). In order to remove the zona pellucida, morulae were briefly incubated in Acid Tyrode’s solution (Sigma) at RT and then washed in M2 medium.
To generate aggregates, embryos were placed in pairs or triplets in aggregation microwells made with an aggregation needle (BLS) on Petri dishes in KSOM medium (LifeGlobal) drops. Drops were overlaid with mineral oil (Nidoil, Nidacon). Single embryos were placed alone as control. KSOM was supplemented with 0.1% BSA (Sigma) to avoid embryos adhering to the plastic. Embryos were cultured at 37°C, 5% CO2 and 90% relative humidity. For MEK inhibition treatment, 1 μM of PD 0325901 (PZ0162, Sigma) was diluted into KSOM. Wild-type embryos were generated by culture in KSOM. 24-hour and 48-hour treated embryos were generated by culture in KSOM with PD 0325901 for 24 hours and 48 hours, respectively. The data were collected over 4 repeat experiments.
To generate aggregates, embryos were placed in pairs or triplets in aggregation microwells made with an aggregation needle (BLS) on Petri dishes in KSOM medium (LifeGlobal) drops. Drops were overlaid with mineral oil (Nidoil, Nidacon). Single embryos were placed alone as control. KSOM was supplemented with 0.1% BSA (Sigma) to avoid embryos adhering to the plastic. Embryos were cultured at 37°C, 5% CO2 and 90% relative humidity. For MEK inhibition treatment, 1 μM of PD 0325901 (PZ0162, Sigma) was diluted into KSOM. Wild-type embryos were generated by culture in KSOM. 24-hour and 48-hour treated embryos were generated by culture in KSOM with PD 0325901 for 24 hours and 48 hours, respectively. The data were collected over 4 repeat experiments.
8
Single-cell isolation from mouse embryos
9
Single-Cell Transcriptomics of ICM Cells
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For E3.5 blastocysts, zona pellucidae were removed using acid tyrode’s solution (Sigma) and embryos subjected to immunosurgery (13 (link), 27 (link)) using 20% anti-mouse whole antiserum (Sigma) in N2B27 at 37 °C, 7% CO2 for 30 min, followed by three rinses in M2 and then 15 min in 20% nonheat inactivated rat serum (made in house) in N2B27 at 37 °C, 7% CO2. After 30 min in fresh N2B27, lysed TE was removed and placed in lysis buffer for genotyping. ICMs were incubated in 0.025% trypsin (Invitrogen) plus 1% chick serum (Sigma) for 5 to 10 min in small drops and dissociated by repetitive pipetting using a small diameter, mouth-controlled, flame-pulled Pasteur pipette. Individual ICM cells were transferred into single-cell lysis buffer and snap frozen on dry ice. Smart-seq2 libraries were prepared as described previously (84 (link)) and sequenced on the Illumina platform in a 125-bp paired-end format.
10
Preimplantation Embryo Isolation and Culture
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