Effectiveness Of Rabbit Anti-14-3-3 Isoform-Specific Antibodies (PAN017, AbD Serotec) To Detect The Proteins In Mouse Oocytes And Eggs

Results Summary

Immunofluorescence microscopy confirms the presence of all seven 14-3-3 isoforms in oocytes and eggs (Figure ​3). The subcellular distributions of the isoforms were found to vary from one isoform to another. For example, 14-3-3ε is expressed uniformly throughout the oocyte with some peripheral accumulation, and absent in the interior of the egg cytoplasm (Figure 3E-F). 14-3-3τ is distributed uniformly in oocytes and eggs, but is particularly absent along the inner nuclear membrane of all oocytes examined (Figure 3I-J). Isoforms 14-3-3β, 14-3-3γ and 14-3-3ζ exhibit a notable peripheral accumulation in oocytes, with a uniform distribution in eggs (Figure 3A-B,3C-D and 3G-H respectively). 14-3-3σ is found to be expressed in higher levels in nuclei of all oocytes studied, as compared to their cytoplasm, where it is uniformly dispersed with some accumulation selectively along one half of the cell (Figure ​3K); however, the 14-3-3σ isoform shows a uniform distribution in eggs (Figure ​3L). 14-3-3η is diffusely dispersed in oocyte with lesser distribution in the germinal vesicle than in the cytoplasm, but attains a uniform punctuate distribution with prominent accumulation in the region of the meiotic spindle in all eggs observed (Figure 3M-N). Control oocytes and eggs incubated in secondary antibody alone displayed little background fluorescence at the same laser intensities and confocal imaging settings used for each immunofluorescence experiment (Figure 3O-P). Localization of 14-3-3η in the meiotic spindle (Figure 3N, Q, R and 3S) suggested a role for the 14-3-3 in spindle assembly or cell cycle control.