Structured Review

Millipore zona pellucida
Electron microscope immunocytochemistry 60 and 90 min after follicular release. (A–C) Oocytes treated with anti-PLC-β1 60 min after follicular release. (A) Gold particles are similarly distributed in the cytoplasm (Cy) and the nucleoplasm (Np); some gold particles are still present on the nuclear envelope (arrow). (B) A large aggregate of gold particles crossing the nuclear envelope. (C) Diffuse staining of the oocyte cortex and cytoplasm; zp, zona <t>pellucida.</t> (D–F) Oocytes treated with anti-PLC-β1 90 min after follicular release. (D) Nucleolus (Nu) is also strongly stained. (E) Gold particles are concentrated in the nucleoplasm and in the IGs; the nuclear envelope (NE) is free of particles. (F) Labeling in the cortex region is noticeable. Magnification, 40,000×. The regions selected in small squares are presented as 2× enlarged inserts.
Zona Pellucida, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Cytoplasmic and Nuclear Phospholipase C-?1 Relocation: Role in Resumption of Meiosis in the Mouse Oocyte"

Article Title: Cytoplasmic and Nuclear Phospholipase C-?1 Relocation: Role in Resumption of Meiosis in the Mouse Oocyte

Journal: Molecular Biology of the Cell

doi:

Electron microscope immunocytochemistry 60 and 90 min after follicular release. (A–C) Oocytes treated with anti-PLC-β1 60 min after follicular release. (A) Gold particles are similarly distributed in the cytoplasm (Cy) and the nucleoplasm (Np); some gold particles are still present on the nuclear envelope (arrow). (B) A large aggregate of gold particles crossing the nuclear envelope. (C) Diffuse staining of the oocyte cortex and cytoplasm; zp, zona pellucida. (D–F) Oocytes treated with anti-PLC-β1 90 min after follicular release. (D) Nucleolus (Nu) is also strongly stained. (E) Gold particles are concentrated in the nucleoplasm and in the IGs; the nuclear envelope (NE) is free of particles. (F) Labeling in the cortex region is noticeable. Magnification, 40,000×. The regions selected in small squares are presented as 2× enlarged inserts.
Figure Legend Snippet: Electron microscope immunocytochemistry 60 and 90 min after follicular release. (A–C) Oocytes treated with anti-PLC-β1 60 min after follicular release. (A) Gold particles are similarly distributed in the cytoplasm (Cy) and the nucleoplasm (Np); some gold particles are still present on the nuclear envelope (arrow). (B) A large aggregate of gold particles crossing the nuclear envelope. (C) Diffuse staining of the oocyte cortex and cytoplasm; zp, zona pellucida. (D–F) Oocytes treated with anti-PLC-β1 90 min after follicular release. (D) Nucleolus (Nu) is also strongly stained. (E) Gold particles are concentrated in the nucleoplasm and in the IGs; the nuclear envelope (NE) is free of particles. (F) Labeling in the cortex region is noticeable. Magnification, 40,000×. The regions selected in small squares are presented as 2× enlarged inserts.

Techniques Used: Microscopy, Immunocytochemistry, Planar Chromatography, Staining, Labeling

2) Product Images from "Hyperactivated sperm motility driven by CatSper2 is required for fertilization"

Article Title: Hyperactivated sperm motility driven by CatSper2 is required for fertilization

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.2136654100

CatSper2 null spermatozoa can fertilize eggs without an extracellular matrix but cannot penetrate a viscous environment. ( a ) Removal of the extracellular matrix of the egg allows CatSper2 null mice to fertilize eggs in vitro ( n = 3 animals for each genotype). Numbers above the bars indicate the total number of eggs examined. ZP, zona pellucida. ( b ) Increased viscosity of the medium does not block wild-type (WT) sperm from swimming but does block the motility of CatSper2 null spermatozoa after incubation in conditions that promote capacitation. The values indicated are expressed as the mean ± SD sperm cell path velocities of the various samples. The open bars represent normal medium, and the filled bars represent medium containing 0.75% (wt/vol) long chain polyacrylamide.
Figure Legend Snippet: CatSper2 null spermatozoa can fertilize eggs without an extracellular matrix but cannot penetrate a viscous environment. ( a ) Removal of the extracellular matrix of the egg allows CatSper2 null mice to fertilize eggs in vitro ( n = 3 animals for each genotype). Numbers above the bars indicate the total number of eggs examined. ZP, zona pellucida. ( b ) Increased viscosity of the medium does not block wild-type (WT) sperm from swimming but does block the motility of CatSper2 null spermatozoa after incubation in conditions that promote capacitation. The values indicated are expressed as the mean ± SD sperm cell path velocities of the various samples. The open bars represent normal medium, and the filled bars represent medium containing 0.75% (wt/vol) long chain polyacrylamide.

Techniques Used: Mouse Assay, In Vitro, Blocking Assay, Incubation

3) Product Images from "Perturbation of the Developmental Potential of Preimplantation Mouse Embryos by Hydroxyurea"

Article Title: Perturbation of the Developmental Potential of Preimplantation Mouse Embryos by Hydroxyurea

Journal: International Journal of Environmental Research and Public Health

doi: 10.3390/ijerph7052033

Normal 2-cell embryo with two equal blastomeres (B), one of two polar bodies (PB) encompassed by an intact zona pellucida (ZP; magnification = 400X).
Figure Legend Snippet: Normal 2-cell embryo with two equal blastomeres (B), one of two polar bodies (PB) encompassed by an intact zona pellucida (ZP; magnification = 400X).

Techniques Used:

Photomicrograph of a blastocyst with an outer cell mass (OCM), inner cell mass (ICM) encompassed by an intact zona pellucida (ZP; Magnification = 400X).
Figure Legend Snippet: Photomicrograph of a blastocyst with an outer cell mass (OCM), inner cell mass (ICM) encompassed by an intact zona pellucida (ZP; Magnification = 400X).

Techniques Used:

4) Product Images from "Role of Fyn kinase in oocyte developmental potential"

Article Title: Role of Fyn kinase in oocyte developmental potential

Journal: Reproduction, fertility, and development

doi: 10.1071/RD09311

Oocyte yield, quality and developmental potential. (a) Oocyte yield was determined by mating age-matched Fyn-null or wild-type (WT) female mice with vasectomised WT males. Oocytes were collected the morning on which vaginal plugs were detected. A separate group of female mice was stimulated with pregnant mare’s serum gonadotropin and human chorionic gonadotrophin before mating. (b) Oocyte size and the thickness of the zona pellucida was measured from images made by Hoffman interference microscopy through the equator of oocytes. (c) The maturation status of oocytes recovered from WT and Fyn-null female mice was determined by confocal fluorescence microscopy following fixation and labelling with ethidium homodimer-2 (EthD-2) to reveal chromosome status. The developmental competence of WT and Fyn-null zygotes produced by mating with fertile WT males was determined by recovering oocytes and zygotes the morning on which vaginal plugs were detected and culturing them to allow development under identical conditions. Oocytes that failed to progress to the two-cell stage by 41 h after mating were fixed, stained with EthD-2 and examined by confocal fluorescence microscopy to determine meiotic and fertilisation status. Immature oocytes were excluded from calculation of the percentage rates of fertilisation and development. Significantly different values are indicated by *, **, ***, or **** (P
Figure Legend Snippet: Oocyte yield, quality and developmental potential. (a) Oocyte yield was determined by mating age-matched Fyn-null or wild-type (WT) female mice with vasectomised WT males. Oocytes were collected the morning on which vaginal plugs were detected. A separate group of female mice was stimulated with pregnant mare’s serum gonadotropin and human chorionic gonadotrophin before mating. (b) Oocyte size and the thickness of the zona pellucida was measured from images made by Hoffman interference microscopy through the equator of oocytes. (c) The maturation status of oocytes recovered from WT and Fyn-null female mice was determined by confocal fluorescence microscopy following fixation and labelling with ethidium homodimer-2 (EthD-2) to reveal chromosome status. The developmental competence of WT and Fyn-null zygotes produced by mating with fertile WT males was determined by recovering oocytes and zygotes the morning on which vaginal plugs were detected and culturing them to allow development under identical conditions. Oocytes that failed to progress to the two-cell stage by 41 h after mating were fixed, stained with EthD-2 and examined by confocal fluorescence microscopy to determine meiotic and fertilisation status. Immature oocytes were excluded from calculation of the percentage rates of fertilisation and development. Significantly different values are indicated by *, **, ***, or **** (P

Techniques Used: Mouse Assay, Microscopy, Fluorescence, Ethidium Homodimer Assay, Produced, Staining

Morphology of Fyn-null oocytes. Oocytes recovered from wild-type (WT) B6;129SF2/J and Fyn-null B6;129S7-Fyn tm1Sor/J (Fyn −/− ) mice were examined by Hoffman modulation contrast. The Fyn-null oocytes contained dense, irregular inclusions. The diameter of the two groups of oocytes did not differ significantly (P = 0.563); however, the thickness of the zona pellucida of Fyn-null oocytes was significantly less than that of WT oocytes (P
Figure Legend Snippet: Morphology of Fyn-null oocytes. Oocytes recovered from wild-type (WT) B6;129SF2/J and Fyn-null B6;129S7-Fyn tm1Sor/J (Fyn −/− ) mice were examined by Hoffman modulation contrast. The Fyn-null oocytes contained dense, irregular inclusions. The diameter of the two groups of oocytes did not differ significantly (P = 0.563); however, the thickness of the zona pellucida of Fyn-null oocytes was significantly less than that of WT oocytes (P

Techniques Used: Mouse Assay

Calcium oscillatory patterns typical of fertilised Fyn-null oocytes. Cumulus- and zona pellucida-free oocytes from nine Fyn-null and five wild-type (WT) female mice were loaded with fura-2, bound to polylysine-coated coverslips and fertilised with WT spermatozoa in vitro so that fertilisation-induced calcium oscillations could be recorded. (a) The typical pattern of calcium oscillations following fertilisation of WT (dashed line) and Fyn-null (solid line) oocytes. (b) The average duration and amplitude of the first calcium oscillation, as well as the total peak number during the first hour after insemination. The total integrated calcium input signal during the first hour after insemination is presented on the far right. The y-axis values show the mean ± s.d.; n values represent the number of recordings analysed from each group. Groups that differ significantly (P
Figure Legend Snippet: Calcium oscillatory patterns typical of fertilised Fyn-null oocytes. Cumulus- and zona pellucida-free oocytes from nine Fyn-null and five wild-type (WT) female mice were loaded with fura-2, bound to polylysine-coated coverslips and fertilised with WT spermatozoa in vitro so that fertilisation-induced calcium oscillations could be recorded. (a) The typical pattern of calcium oscillations following fertilisation of WT (dashed line) and Fyn-null (solid line) oocytes. (b) The average duration and amplitude of the first calcium oscillation, as well as the total peak number during the first hour after insemination. The total integrated calcium input signal during the first hour after insemination is presented on the far right. The y-axis values show the mean ± s.d.; n values represent the number of recordings analysed from each group. Groups that differ significantly (P

Techniques Used: Mouse Assay, In Vitro

5) Product Images from "Self-organisation of the human embryo in the absence of maternal tissues"

Article Title: Self-organisation of the human embryo in the absence of maternal tissues

Journal: Nature cell biology

doi: 10.1038/ncb3347

Establishment of an in vitro system to study human implantation and early post-implantation morphogenesis. Human embryos were thawed and cultured until the blastocyst stage (day 5-6 of development). The zona pellucida was removed and embryos were transferred to plates in IVC1 medium for imaging. On the second day of culture, medium was changed for IVC2 with 30% KnockOut Serum Replacement (KSR) (b, c) or 20% human cord serum (HCS) ( d) . Shown are representative bright field images of human blastocysts developing in vitro until day 12-13. All scale bars, 100 μm. These data involved the assessment of a total of 5 embryos collected across 3 experiments, out of which 3 showed a correct development.
Figure Legend Snippet: Establishment of an in vitro system to study human implantation and early post-implantation morphogenesis. Human embryos were thawed and cultured until the blastocyst stage (day 5-6 of development). The zona pellucida was removed and embryos were transferred to plates in IVC1 medium for imaging. On the second day of culture, medium was changed for IVC2 with 30% KnockOut Serum Replacement (KSR) (b, c) or 20% human cord serum (HCS) ( d) . Shown are representative bright field images of human blastocysts developing in vitro until day 12-13. All scale bars, 100 μm. These data involved the assessment of a total of 5 embryos collected across 3 experiments, out of which 3 showed a correct development.

Techniques Used: In Vitro, Cell Culture, Imaging, Knock-Out

Related Articles

Isolation:

Article Title: Dynamic expression of chromatin modifiers during developmental transitions in mouse preimplantation embryos
Article Snippet: Embryos were cultured in KSOMaa (Milipore, MR-106-D) in a low oxygen air chamber. .. Single blastomeres were isolated by first removing the Zona pellucida with Proteinase (Sigma, P5147-1 G, 5 mg/ml in M2 medium), followed by separating the cells with Trypsine (Trypsine-EDTA, T4049 Sigma). .. Single cells were picked with the help of a mouth pipette and a finely pulled glass capillary.

other:

Article Title: Effect of Substrate Stiffness on Early Mouse Embryo Development
Article Snippet: Removal of the Zona Pellucida The zona-pellucida (ZP) was removed by immersing the embryos in acid Tyrode’s solution (Sigma), followed by immediate washing in KSOM .

TUNEL Assay:

Article Title: HIPPO signaling resolves embryonic cell fate conflicts during establishment of pluripotency in vivo
Article Snippet: TUNEL assay Embryos were fixed, permeabilized, and blocked as described for immunofluorescence. .. Zonae pellucida were removed using Tyrode’s Acid treatment prior to performing the TUNEL assay (In Situ Cell Death Detection Kit, Fluorescein, Millipore-Sigma). .. Embryos were incubated in 200 µl of a 1:10 dilution of enzyme in label solution for 2 hr at 37°C.

In Situ:

Article Title: HIPPO signaling resolves embryonic cell fate conflicts during establishment of pluripotency in vivo
Article Snippet: TUNEL assay Embryos were fixed, permeabilized, and blocked as described for immunofluorescence. .. Zonae pellucida were removed using Tyrode’s Acid treatment prior to performing the TUNEL assay (In Situ Cell Death Detection Kit, Fluorescein, Millipore-Sigma). .. Embryos were incubated in 200 µl of a 1:10 dilution of enzyme in label solution for 2 hr at 37°C.

Incubation:

Article Title: Nuclei size in relation to nuclear status and aneuploidy rate for 13 chromosomes in donated four cells embryos
Article Snippet: .. At 50 h (±2 h) after oocyte aspiration the embryos were transferred for about 1 min to culture medium containing pronase (5 mg/ml; Sigma, St. Louis, MO, USA) to dissolve the zona pellucida followed by incubation in Ca2+ /Mg2+ -free medium (EB-10; Vitrolife, Gothenburg, Sweden) for 1 to 4 min until segregation of the individual blastomeres. ..

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    Millipore zona pellucida free
    Spermiograms and in vitro fertilization assays. Spermiograms from PCI +/+ ( a ) and from PCI +/– ( b ) were normal, whereas in spermiograms from PCI –/– mice ( c ) immature and malformed cells were prevalent. ( d ) In in vitro fertilization assays, sperm obtained from PCI +/– mice were able to bind and subsequently fertilize PCI +/+ oocytes. Sperm obtained from PCI –/– males failed to fertilize zona <t>pellucida–containing</t> ( e ) or zona pellucida–free ( f ) oocytes.
    Zona Pellucida Free, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/zona pellucida free/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    zona pellucida free - by Bioz Stars, 2021-04
    97/100 stars
      Buy from Supplier

    97
    Millipore in vitro cleavage assay zonae pellucidae
    Cleavage of ZP2 by ovastacin. (A) A schematic of mouse ZP2 with eight diacidic motifs (triangles), the initial cleavage site of ovastacin, the binding sites of mAbs IE-3 and M2c.2 N terminal and C terminal to the initial cleavage site, and the localization of the single N -glycan in the N terminus. The potential size of peptides resulting from cleavage of the N-terminal fragment with ovastacin detected by IE-3 is shown on the bottom. (B) Immunoblot of eggs (15) or two-cell embryos (15) from normal and Astl Null females stained with IE-3 mAb that detects N-terminal fragments of ZP2. The top bands (120 kD) are intact ZP2, and bottom bands are a heterogeneous mixture of cleaved N-terminal fragments. Arrows indicate the four peptides detected on the immunoblot probed with IE-3. (C) Isolated <t>zonae</t> <t>pellucidae</t> (15–20) were incubated with partially purified recombinant ovastacin (Ovastacin Rec ) at 37°C for 0, 2, 4, and 8 h. (D) Same as in C but with insect cell supernatant lacking (control [Ctrl]) or containing (Ovst Rec ) recombinant ovastacin after 4 h of incubation with zonae pellucidae from normal (Norm) or Astl Null eggs. (C and D) Cleavage was detected by immunoblotting with M2c.2 to detect the ZP2 C-terminal fragment.
    In Vitro Cleavage Assay Zonae Pellucidae, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/in vitro cleavage assay zonae pellucidae/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    in vitro cleavage assay zonae pellucidae - by Bioz Stars, 2021-04
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    97
    Millipore zona pellucida
    Normal 2-cell embryo with two equal blastomeres (B), one of two polar bodies (PB) encompassed by an intact zona <t>pellucida</t> (ZP; magnification = 400X).
    Zona Pellucida, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/zona pellucida/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    zona pellucida - by Bioz Stars, 2021-04
    97/100 stars
      Buy from Supplier

    Image Search Results


    Spermiograms and in vitro fertilization assays. Spermiograms from PCI +/+ ( a ) and from PCI +/– ( b ) were normal, whereas in spermiograms from PCI –/– mice ( c ) immature and malformed cells were prevalent. ( d ) In in vitro fertilization assays, sperm obtained from PCI +/– mice were able to bind and subsequently fertilize PCI +/+ oocytes. Sperm obtained from PCI –/– males failed to fertilize zona pellucida–containing ( e ) or zona pellucida–free ( f ) oocytes.

    Journal: The Journal of Clinical Investigation

    Article Title: Disruption of the protein C inhibitor gene results in impaired spermatogenesis and male infertility

    doi:

    Figure Lengend Snippet: Spermiograms and in vitro fertilization assays. Spermiograms from PCI +/+ ( a ) and from PCI +/– ( b ) were normal, whereas in spermiograms from PCI –/– mice ( c ) immature and malformed cells were prevalent. ( d ) In in vitro fertilization assays, sperm obtained from PCI +/– mice were able to bind and subsequently fertilize PCI +/+ oocytes. Sperm obtained from PCI –/– males failed to fertilize zona pellucida–containing ( e ) or zona pellucida–free ( f ) oocytes.

    Article Snippet: These oocytes were either surrounded by cumulus cells, cumulus cell–free (removed by treatment with 0.1% hyaluronidase), or zona pellucida–free (removed by treatment with 0.1% proteinase K; Sigma-Aldrich).

    Techniques: In Vitro, Mouse Assay

    Cleavage of ZP2 by ovastacin. (A) A schematic of mouse ZP2 with eight diacidic motifs (triangles), the initial cleavage site of ovastacin, the binding sites of mAbs IE-3 and M2c.2 N terminal and C terminal to the initial cleavage site, and the localization of the single N -glycan in the N terminus. The potential size of peptides resulting from cleavage of the N-terminal fragment with ovastacin detected by IE-3 is shown on the bottom. (B) Immunoblot of eggs (15) or two-cell embryos (15) from normal and Astl Null females stained with IE-3 mAb that detects N-terminal fragments of ZP2. The top bands (120 kD) are intact ZP2, and bottom bands are a heterogeneous mixture of cleaved N-terminal fragments. Arrows indicate the four peptides detected on the immunoblot probed with IE-3. (C) Isolated zonae pellucidae (15–20) were incubated with partially purified recombinant ovastacin (Ovastacin Rec ) at 37°C for 0, 2, 4, and 8 h. (D) Same as in C but with insect cell supernatant lacking (control [Ctrl]) or containing (Ovst Rec ) recombinant ovastacin after 4 h of incubation with zonae pellucidae from normal (Norm) or Astl Null eggs. (C and D) Cleavage was detected by immunoblotting with M2c.2 to detect the ZP2 C-terminal fragment.

    Journal: The Journal of Cell Biology

    Article Title: Ovastacin, a cortical granule protease, cleaves ZP2 in the zona pellucida to prevent polyspermy

    doi: 10.1083/jcb.201112094

    Figure Lengend Snippet: Cleavage of ZP2 by ovastacin. (A) A schematic of mouse ZP2 with eight diacidic motifs (triangles), the initial cleavage site of ovastacin, the binding sites of mAbs IE-3 and M2c.2 N terminal and C terminal to the initial cleavage site, and the localization of the single N -glycan in the N terminus. The potential size of peptides resulting from cleavage of the N-terminal fragment with ovastacin detected by IE-3 is shown on the bottom. (B) Immunoblot of eggs (15) or two-cell embryos (15) from normal and Astl Null females stained with IE-3 mAb that detects N-terminal fragments of ZP2. The top bands (120 kD) are intact ZP2, and bottom bands are a heterogeneous mixture of cleaved N-terminal fragments. Arrows indicate the four peptides detected on the immunoblot probed with IE-3. (C) Isolated zonae pellucidae (15–20) were incubated with partially purified recombinant ovastacin (Ovastacin Rec ) at 37°C for 0, 2, 4, and 8 h. (D) Same as in C but with insect cell supernatant lacking (control [Ctrl]) or containing (Ovst Rec ) recombinant ovastacin after 4 h of incubation with zonae pellucidae from normal (Norm) or Astl Null eggs. (C and D) Cleavage was detected by immunoblotting with M2c.2 to detect the ZP2 C-terminal fragment.

    Article Snippet: In vitro cleavage assay Zonae pellucidae were isolated from 150 oocytes by freeze thawing four times in 100 µl PBS, pH 7.4, 0.1% IGEPAL CA-630 (Sigma-Aldrich), and 0.5 M NaCl.

    Techniques: Binding Assay, Staining, Isolation, Incubation, Purification, Recombinant

    Normal 2-cell embryo with two equal blastomeres (B), one of two polar bodies (PB) encompassed by an intact zona pellucida (ZP; magnification = 400X).

    Journal: International Journal of Environmental Research and Public Health

    Article Title: Perturbation of the Developmental Potential of Preimplantation Mouse Embryos by Hydroxyurea

    doi: 10.3390/ijerph7052033

    Figure Lengend Snippet: Normal 2-cell embryo with two equal blastomeres (B), one of two polar bodies (PB) encompassed by an intact zona pellucida (ZP; magnification = 400X).

    Article Snippet: Normal 2-cell mouse embryos, each defined as an embryo with two blastomeres of equal size with two polar bodies in the perivitelline space and encompassed by an intact zona pellucida were pooled among mice within treatment and washed in several droplets of WM containing 1% BSA (Sigma Chemical Co., St. Louis, MO).

    Techniques:

    Photomicrograph of a blastocyst with an outer cell mass (OCM), inner cell mass (ICM) encompassed by an intact zona pellucida (ZP; Magnification = 400X).

    Journal: International Journal of Environmental Research and Public Health

    Article Title: Perturbation of the Developmental Potential of Preimplantation Mouse Embryos by Hydroxyurea

    doi: 10.3390/ijerph7052033

    Figure Lengend Snippet: Photomicrograph of a blastocyst with an outer cell mass (OCM), inner cell mass (ICM) encompassed by an intact zona pellucida (ZP; Magnification = 400X).

    Article Snippet: Normal 2-cell mouse embryos, each defined as an embryo with two blastomeres of equal size with two polar bodies in the perivitelline space and encompassed by an intact zona pellucida were pooled among mice within treatment and washed in several droplets of WM containing 1% BSA (Sigma Chemical Co., St. Louis, MO).

    Techniques: