Computational protocol: The Role of Glucose Metabolism on Porcine Oocyte Cytoplasmic Maturation and Its Possible Mechanisms

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Protocol publication

[…] We performed the procedures as previously described []. Briefly, blastocysts were fixed for 30 minutes in 3.7% paraformaldehyde prepared in phosphate buffered saline with 0.1% (w/v) polyvinyl alcohol (PBS-PVA). Afterwards, they were washed with PBS, and then transferred to 0.3% Triton X-100 for 1 h. Thereafter, they were washed twice with PBS, and incubated in the dark for 1h after the addition of fluorescein-conjugated dUTP and terminal deoxynucleotidyl transferase. After a 1 h incubation with 10 μg/mL Hoechst 33342 and 50 mg/mL RNase A, the blastocysts were examined using a laser scanning confocal microscope. The total number of cells and the number of apoptotic cells were determined using ImageJ software (Public Domain). [...] All experimental results were analyzed using IBM SPSS Statistics v19 (IBM, Armonk, NY, USA). One-way ANOVA and Chi-square test were performed to evaluate the statistical significance of differences, all percentage data were subjected to arc sine transformation before statistical analysis. A p-value lower than 0.05 was considered statistically significant. […]

Pipeline specifications

Software tools ImageJ, SPSS
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Chemicals Adenosine Triphosphate, Dehydroepiandrosterone, Glucose, NADP, Pentosephosphates, Pyruvic Acid