Subcellular Localization of Cytoplasmic Lattice-Associated Proteins Is Dependent upon Fixation and Processing ProceduresReportar como inadecuado




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We and others have recently demonstrated by immuno-EM and mutation analysis that two oocyte-restricted maternal effect genes, PADI6 and MATER, localize, in part, to the oocyte cytoplasmic lattices CPLs. During these ongoing studies, however, we found that the localization of these factors by confocal immunofluorescence IF analysis can vary dramatically depending upon how the oocytes and embryos are processed, with the localization pattern sometimes appearing more uniformly cytoplasmic while at other times appearing to be primarily cortical. We set out to better understand this differential staining pattern by testing a range of IF protocol parameters, changing mainly time and temperature conditions of the primary antibody solution incubation, as well as fixation methods. We found by confocal IF whole mount analysis that PADI6 and MATER localization in germinal vesicle stage oocytes is mainly cytoplasmic when the oocytes are fixed and then incubated with primary antibodies at room temperature for 1 hour, while the localization of these factors is largely limited to the cortex when the oocytes are fixed and incubated in primary antibody at 4°C overnight. We then probed sections of fixed-embedded ovaries and isolated two-cell embryos with specific antibodies and found that, under these conditions, PADI6 and MATER were again primarily cytoplasmically localized, although the staining for these factors is slightly more cortical at the two-cell stage. Taken together, our results suggest that the localization of CPL-associated proteins by confocal IF is particularly affected by processing conditions. Further, based on our current observations, it appears that PADI6 and MATER are primarily distributed throughout the cytoplasm as opposed to the oocyte subcortex.



Autor: Eric Morency, Lynne Anguish, Scott Coonrod

Fuente: http://plos.srce.hr/



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