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Acid tyrode

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Acid Tyrodes is a laboratory solution used in various cell and tissue culture applications. It serves as a balanced salt solution, maintaining the pH and osmotic environment suitable for sustaining cells and tissues in an in vitro setting. The solution contains a specific combination of inorganic salts, glucose, and other components that mimic the composition of physiological fluids.

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Lab products found in correlation

Smarter ultra low input rna cdna preparation kit, by Takara Bio (2 mentions) Hiseq 2500 sequencer, by Illumina (2 mentions) Nextera xt dna library preparation kit, by Illumina (2 mentions) A1 laser scanning confocal microscope, by Nikon (2 mentions) Vvl fitc, by Vector Laboratories (2 mentions) M2 medium, by Merck Group (2 mentions) Ksom hepes buffer, by Merck Group (2 mentions) High capacity reverse transcription kit, by Thermo Fisher Scientific (2 mentions) Ksom aa, by Merck Group (2 mentions) Rneasy micro kit, by Qiagen (2 mentions) Chemidoc, by Bio-Rad (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)

Close spelling variants of this product

Acid Tyrode solution Tyrode’s acid Acid Tyrode’s solution

7 protocols using acid tyrode

1

RNA-seq of Dux F2 embryos

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For embryos collected for RNA-seq (i.e., Dux F2 × F2), IVF was performed as described above except that the micro-injection steps were omitted. Late 1-cell and late 2-cell were collected at ~12 and ~30 hpi, respectively. For each biological replicate, 11–13 embryos were pooled for RNA-seq analyses. Specifically, the embryos were briefly incubated in Acid Tyrode (Millipore) to remove zona pellucida and then washed three times in 0.2% BSA/PBS prior for library construction.
RNA-seq libraries were prepared as previously described 25 . Briefly, SMARTer Ultra Low Input RNA cDNA preparation kit (Clontech, 643890) was used for reverse transcription and cDNA amplification (11 cycles). cDNA were then fragmented, adaptor-ligated and amplified using Nextera XT DNA Library Preparation Kit (Illumina) according to the manufacturer’s instructions. Single-end 100-bp sequencing was performed on a HiSeq 2500 sequencer (Illumina). The summary of the generated datasets can be found in Supplementary Table 5.
Chen Z, & Zhang Y. (2019). Loss of DUX causes minor defects in zygotic genome activation and is compatible with mouse development. Nature genetics, 51(6), 947-951.
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2

RNA-seq of Dux F2 embryos

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For embryos collected for RNA-seq (i.e., Dux F2 × F2), IVF was performed as described above except that the micro-injection steps were omitted. Late 1-cell and late 2-cell were collected at ~12 and ~30 hpi, respectively. For each biological replicate, 11–13 embryos were pooled for RNA-seq analyses. Specifically, the embryos were briefly incubated in Acid Tyrode (Millipore) to remove zona pellucida and then washed three times in 0.2% BSA/PBS prior for library construction.
RNA-seq libraries were prepared as previously described 25 . Briefly, SMARTer Ultra Low Input RNA cDNA preparation kit (Clontech, 643890) was used for reverse transcription and cDNA amplification (11 cycles). cDNA were then fragmented, adaptor-ligated and amplified using Nextera XT DNA Library Preparation Kit (Illumina) according to the manufacturer’s instructions. Single-end 100-bp sequencing was performed on a HiSeq 2500 sequencer (Illumina). The summary of the generated datasets can be found in Supplementary Table 5.
Chen Z, & Zhang Y. (2019). Loss of DUX causes minor defects in zygotic genome activation and is compatible with mouse development. Nature genetics, 51(6), 947-951.
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3

Immunofluorescence Staining of Embryos

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The zona pellucida was removed by Acid Tyrode (Millipore). The embryos were fixed with 2% paraformaldehyde (PFA) in PBS containing 0.1% polyvinyl alcohol (PBS-PVA) for 15 min at room temperature (RT), which was followed by permeabilization with 0.25% Triton X-100 in PBS-PVA for 15 min at RT. The embryos were blocked in 1% BSA in PBS-PVA for 1 hour at RT. After blocking, embryos were then incubated with primary antibody overnight at 4°C. The embryos were washed in PBS-PVA and incubated with Alexa Fluor 546 IgG secondary antibodies (1:500; Life Technologies) for 1 h at RT. After the embryos were washed with PBS-PVA and attached to cover slides, the nuclei were stained with DAPI (Vector Laboratories, California, USA). Antibodies for Oct4-C10 and Oct4-A9 were used in this study (1:500; Santa Cruz Biotechnology, Dallas, TX, USA). Imaging of the embryos was performed using Zeiss LSM 510 META laser scanning confocal system.
Mitani A., Fukuda A., Miyashita T., Umezawa A, & Akutsu H. (2017). The serine 106 residue within the N-terminal transactivation domain is crucial for Oct4 function in mice. Zygote (Cambridge, England), 25(2).
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4

Blastocyst Isolation and Imaging

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Wild-type 129/SvEv mice were euthanized 3.5 days post-mating. The uterus was isolated and washed in KSOM-HEPES buffer (Milli-pore Sigma). The utero-tubule junctions were cut and the uterine horns were flushed using a 25 gauge needle filled with 0.4 mL of M2 medium (Millipore Sigma). Using a mouth controlled Pasteur pipette, blastocysts were collected and transferred into a drop of PBS. Blastocysts were hatched by serial passage through drops of Acid Tyrodes (Millipore Sigma). Hatched blastocysts were transferred into drops of PBS, and then fixed in 3% paraformaldehyde and VVL immunofluorescence staining was performed as described under Vicia villosa lectin assays. Blastocyst images were obtained using EVOS epifluorescence and Nikon A1 laser scanning confocal microscope. Immunostaining of the trophectoderm of intact, infected blastocysts was performed 48 hours post-infection. The shControl or shGalnt3 infected blastocysts were fixed with 3% paraformaldehyde and blocked for 30 min using 1X carbofree block. Blastocysts were stained for 1 hour using VVL-FITC (Vector Laboratories) at room temperature. The VVL-FITC stained blastocysts were imaged at 20X using EVOS microscopy.
Raghu D., Mobley R.J., Shendy N.A., Perry C.H, & Abell A.N. (2019). GALNT3 Maintains the Epithelial State in Trophoblast Stem Cells. Cell reports, 26(13), 3684-3697.e7.
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5

Blastocyst Infection and Trophoblast Analysis

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Wild-type blastocysts were isolated from timed matings of pure 129/SvEv males with females in the pure 129/SvEv and 129/SvEv/C57B6 mixed backgrounds. Mid-stage blastocysts were hatched by transfer into drops of Acid-tyrodes (Millipore Sigma) for two min. The trophectoderm layer of the hatched, intact blastocysts was infected as previously described (Georgiades et al., 2007 (link)). Briefly, 100 μL drops of lentiviruses encoding shControl or shGalnt3 resuspended in KSOM-AA (Millipore Sigma) were used to infect hatched blastocysts for 6 hours. Post infection, blastocysts were transferred into KSOM-AA for 24 hours. Attachment of the blastocysts was assessed after transfer of infected blastocysts into 70% TS medium supplemented with FGF4.
For measurement of puromycin resistance gene expression, RNA was isolated five days post infection from attached and differentiated trophoblast cells in shControl and shGalnt3 using RNeasy micro kit (QIAGEN). High-Capacity reverse transcription kit (Thermo Fisher Scientific) was used to prepare cDNA. PCR reactions for puromycin resistance gene encoded by the lentiviruses were performed. PCR products were run on a 2% agarose gels and imaged using Bio-Rad ChemiDoc.
Raghu D., Mobley R.J., Shendy N.A., Perry C.H, & Abell A.N. (2019). GALNT3 Maintains the Epithelial State in Trophoblast Stem Cells. Cell reports, 26(13), 3684-3697.e7.
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6

Blastocyst Infection and Trophoblast Analysis

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Wild-type blastocysts were isolated from timed matings of pure 129/SvEv males with females in the pure 129/SvEv and 129/SvEv/C57B6 mixed backgrounds. Mid-stage blastocysts were hatched by transfer into drops of Acid-tyrodes (Millipore Sigma) for two min. The trophectoderm layer of the hatched, intact blastocysts was infected as previously described (Georgiades et al., 2007 (link)). Briefly, 100 μL drops of lentiviruses encoding shControl or shGalnt3 resuspended in KSOM-AA (Millipore Sigma) were used to infect hatched blastocysts for 6 hours. Post infection, blastocysts were transferred into KSOM-AA for 24 hours. Attachment of the blastocysts was assessed after transfer of infected blastocysts into 70% TS medium supplemented with FGF4.
For measurement of puromycin resistance gene expression, RNA was isolated five days post infection from attached and differentiated trophoblast cells in shControl and shGalnt3 using RNeasy micro kit (QIAGEN). High-Capacity reverse transcription kit (Thermo Fisher Scientific) was used to prepare cDNA. PCR reactions for puromycin resistance gene encoded by the lentiviruses were performed. PCR products were run on a 2% agarose gels and imaged using Bio-Rad ChemiDoc.
Raghu D., Mobley R.J., Shendy N.A., Perry C.H, & Abell A.N. (2019). GALNT3 Maintains the Epithelial State in Trophoblast Stem Cells. Cell reports, 26(13), 3684-3697.e7.
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7

Blastocyst Isolation and Imaging

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Wild-type 129/SvEv mice were euthanized 3.5 days post-mating. The uterus was isolated and washed in KSOM-HEPES buffer (Milli-pore Sigma). The utero-tubule junctions were cut and the uterine horns were flushed using a 25 gauge needle filled with 0.4 mL of M2 medium (Millipore Sigma). Using a mouth controlled Pasteur pipette, blastocysts were collected and transferred into a drop of PBS. Blastocysts were hatched by serial passage through drops of Acid Tyrodes (Millipore Sigma). Hatched blastocysts were transferred into drops of PBS, and then fixed in 3% paraformaldehyde and VVL immunofluorescence staining was performed as described under Vicia villosa lectin assays. Blastocyst images were obtained using EVOS epifluorescence and Nikon A1 laser scanning confocal microscope. Immunostaining of the trophectoderm of intact, infected blastocysts was performed 48 hours post-infection. The shControl or shGalnt3 infected blastocysts were fixed with 3% paraformaldehyde and blocked for 30 min using 1X carbofree block. Blastocysts were stained for 1 hour using VVL-FITC (Vector Laboratories) at room temperature. The VVL-FITC stained blastocysts were imaged at 20X using EVOS microscopy.
Raghu D., Mobley R.J., Shendy N.A., Perry C.H, & Abell A.N. (2019). GALNT3 Maintains the Epithelial State in Trophoblast Stem Cells. Cell reports, 26(13), 3684-3697.e7.
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