zp2 antibody atcc ie3 crl  (ATCC)

Journal: bioRxiv

Article Title: A novel and critical role of the intracellular Zona Pellucida protein 2 (ZP2) for blastocyst formation in mice

doi: 10.64898/2025.12.12.692802

Figure Lengend Snippet: Mass spectrometric (LC-MS/MS) quantification of ZP protein levels during mouse pre-implantation development. Mouse embryos were collected at consecutive stages of pre-implantation development in vitro after culture in KSOM(aa) medium, and subjected to LC-MS/MS. Zona pellucida was either left in place (zona intact) or removed (zona free). Shown are the relative iBAQ (riBAQ) values of protein abundance for (A) ZP proteins and (B) housekeeping proteins. The color coding, indicative of expression level, was applied in each row independently of the other rows. The raw proteomic data are available in PRIDE repository with accession number PXD056725 and are provided in simplified form in Supplementary Table S1. Abbreviations: riBAQ, relative intensity Based Absolute Quantification [ , ]; LC-MS/MS, Liquid Chromatography with tandem mass spectrometry. Abbreviations: iBAQ, intensity-based absolute quantification; LC-MS/MS, Liquid Chromatography with tandem mass spectrometry; MII, metaphase II oocyte.

Article Snippet: For the direct and indirect IF, the primary ZP2 antibody ATCC IE3 CRL-2463 was applied at 1:200 and incubated overnight at 4 °C.

Techniques: Liquid Chromatography with Mass Spectroscopy, In Vitro, Quantitative Proteomics, Expressing, Liquid Chromatography, Mass Spectrometry

Journal: bioRxiv

Article Title: A novel and critical role of the intracellular Zona Pellucida protein 2 (ZP2) for blastocyst formation in mice

doi: 10.64898/2025.12.12.692802

Figure Lengend Snippet: Immunofluorescence profiling of ZP2 protein during mouse pre-implantation development. (A) Developmental series of embryos cultured in vitro and subjected to indirect IF. (B) Developmental series of embryos cultured in vitro and subjected to direct IF. (C) Developmental series of embryos obtained fresh from the genital tract without in vitro culture and examined by indirect IF. Numbers shown in the top left corner of each diagram indicate the total numbers of embryos examined across all stages. The integrated density values of the ZP2 IF intensities were quantified using Image-J, normalized to the 1-cell stage (set to 1) and plotted by stage of development. Each point corresponds to the normalized integrated density one embryo. Each point corresponds to the normalized integrated density one embryo. All normalized integrated density values are provided in Supplementary Table S2. P-values were calculated with Wilcoxon test. Abbreviations: IF, immunofluorescence; MII, metaphase II (oocyte).

Article Snippet: For the direct and indirect IF, the primary ZP2 antibody ATCC IE3 CRL-2463 was applied at 1:200 and incubated overnight at 4 °C.

Techniques: Immunofluorescence, Cell Culture, In Vitro

Journal: bioRxiv

Article Title: A novel and critical role of the intracellular Zona Pellucida protein 2 (ZP2) for blastocyst formation in mice

doi: 10.64898/2025.12.12.692802

Figure Lengend Snippet: Intracellular increase of ZP2 protein abundance is contributed by de novo protein synthesis. (A). Developmental progression of zygotes microinjected with various concentrations of morpholino targeting Zp2 mRNA (treatment) or morpholino targeting an irrelevant sequence (GFP; negative control). Irrespective of the morpholino target, zygotes can hardly progress to the 8-cell stage at morpholino concentrations higher than 0.1 mM. Although this is suggestive of unspecific toxicity of morpholino no matter which target, it should be noted that targeting ZP2 is more detrimental than targeting GFP, as seen from the fact that the green bars in (A) are more extended to the right than the blue bars. At morpholino concentration of 0.05 mM both treatment and control zygotes progressed to blastocyst, however at rates substantially lower than 100% also in the negative control, suggestive of unspecific toxicity. At morpholino concentration of 0.01 mM majority of zygotes progressed to blastocyst in both treatment and control groups, indicating that this concentration is suited to test for gene-specific translation-blocking effects without incurring unspecific toxicity. Numbers of zygotes (n) microinjected with anti-ZP2 morpholino (mM concentration): 34 (0.01), 32 (0.05), 27 (0.1), 24 (0.5), 46 (1.0). Numbers of zygotes microinjected with control morpholino (mM concentration): 36 (0.01), 33 (0.05), 22 (0.1), 22 (0.5), 32 (1.0). (B). Zygotes microinjected with 0.01 mM morpholino were sampled at the 8-cell stage and subjected to ZP2 immunofluorescence using antibody ATCC cat.no. IE-3 – CRL-246. The immunofluorescence intensity values are provided in Supplementary Table S3. Immunofluorescence intensity quantification revealed that ZP2 intensity was reduced by ∼ 20% after translational inhibition of ZP2 mRNA compared to that of negative control. Representative images of the 8-cell embryos are shown after immunostaining with anti ZP2 (ATCC cat.no. IE-3 – CRL-246) revealed by secondary antibody. P-value was calculated with Wilcoxon test. *, <0.05. Abbreviations: IntDen, Integrated density of immunofluorescence signal (intensity x area).

Article Snippet: For the direct and indirect IF, the primary ZP2 antibody ATCC IE3 CRL-2463 was applied at 1:200 and incubated overnight at 4 °C.

Techniques: Quantitative Proteomics, Sequencing, Negative Control, Concentration Assay, Control, Blocking Assay, Immunofluorescence, Inhibition, Immunostaining

Journal: bioRxiv

Article Title: A novel and critical role of the intracellular Zona Pellucida protein 2 (ZP2) for blastocyst formation in mice

doi: 10.64898/2025.12.12.692802

Figure Lengend Snippet: Intracellular ZP2 is required for the morula-to-blastocyst transition and healthy blastocyst formation. Zygotes were microinjected with protein KD reagents and cultured further. (A). At the morula stage (72h post fertilization), ZP2-KD embryos lagged behind in cavitation compared to mock KD and MC embryos. Numbers underneath the box plots are total number of embryos across the stages. The raw data of the developmental rates are provided in Supplementary Table S4. (B) At the blastocyst stage (96h post fertilization), ZP2-KD embryos were deficient in trophectoderm and primitive endoderm compared to mock KD and MC embryos. Numbers underneath the pie charts refer to the blastocysts imaged after triple immunofluorescence. The raw data of the cell counts are provided in Supplementary Table S5. (C) When examined for their ability to develop further, ZP2-KD blastocysts were less able of escaping the zona pellucida and forming outgrowths when plated onto a fibroblast layer in vitro ; in vivo they were less able to develop to term when transferred to pseudopregnant uterus, compared to mock KD and MC blastocysts. Numbers underneath the bar plots are the blastocysts plated on feeders (implantation assay in vitro ) or the fetuses obtained from the blastocysts transferred to uterus (embryo transfer in vivo ). The raw data of the outgrowth and embryo transfers are provided in Supplementary Tables S6 and S7, respectively. P-values were calculated with Wilcoxon test. Abbreviations: ZP2 KD, ZP2 knockdown; mock KD, mock knockdown; MC, micromanipulation control.

Article Snippet: For the direct and indirect IF, the primary ZP2 antibody ATCC IE3 CRL-2463 was applied at 1:200 and incubated overnight at 4 °C.

Techniques: Cell Culture, Immunofluorescence, In Vitro, In Vivo, Knockdown, Micromanipulation, Control

Journal: bioRxiv

Article Title: A novel and critical role of the intracellular Zona Pellucida protein 2 (ZP2) for blastocyst formation in mice

doi: 10.64898/2025.12.12.692802

Figure Lengend Snippet: Transcriptomic correlates of unhealthy blastocyst formation after KD of ZP2. Zygotes were microinjected with protein KD reagents and cultured to blastocyst stage. (A) Hierarchical clustering of the 12920 protein-coding mRNAs detected in all samples. (B) Principal component analysis of the samples based on the protein-coding mRNAs. (C) Volcano plots of the mRNAs differentially expressed between ZP2 KD and MC and between mock KD and MC, highlighting the mRNAs that satisfy the criteria of both p<0.05 (Student’s t-test) and |fold change|ti2. The numbers in the colored areas indicate the respective differentially expressed mRNAs. (D) Venn diagram identification of mRNAs differentially exclusively in ZP2 KD and mock KD. Overrepresentation analysis of the mRNAs differentially expressed exclusively in ZP2 KD and mock KD in the ontology “Biological process”. Abbreviations: ZP2 KD, ZP2 knockdown; mock KD, mock knockdown; MC, micromanipulation control.

Article Snippet: For the direct and indirect IF, the primary ZP2 antibody ATCC IE3 CRL-2463 was applied at 1:200 and incubated overnight at 4 °C.

Techniques: Cell Culture, Knockdown, Micromanipulation, Control

Journal: bioRxiv

Article Title: A novel and critical role of the intracellular Zona Pellucida protein 2 (ZP2) for blastocyst formation in mice

doi: 10.64898/2025.12.12.692802

Figure Lengend Snippet: Proteomic correlates of unhealthy blastocyst formation after KD of ZP2. Zygotes were microinjected with protein KD reagents and cultured to later morula stage. (A) Hierarchical clustering of the 2401 proteins detected in all samples. (B) Principal component analysis of the samples based on the 2401 proteins. (C) Volcano plots of the proteins differentially expressed between ZP2 KD and MC and between mock KD and MC, highlighting the proteins that satisfy the criteria of both p<0.05 (Student’s t-test) and |fold change|ti2. The numbers in the colored areas indicate the respective differentially expressed proteins. (D) Venn diagram identification of proteins differentially exclusively in ZP2 KD and mock KD and mock. Overrepresentation analysis of the proteins differentially expressed exclusively in ZP2 KD and mock KD in the ontology “Biological process”. Abbreviations: ZP2 KD, ZP2 knockdown; mock KD, mock knockdown; MC, micromanipulation control.

Article Snippet: For the direct and indirect IF, the primary ZP2 antibody ATCC IE3 CRL-2463 was applied at 1:200 and incubated overnight at 4 °C.

Techniques: Cell Culture, Knockdown, Micromanipulation, Control