Subcellular localization of cytoplasmic lattice-associated proteins is dependent upon fixation and processing procedures.
Subcellular localization of cytoplasmic lattice-associated proteins is dependent upon fixation and processing procedures.
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DOI：
10.1371/journal.pone.0017226
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发表时间：
2011-02-16
期刊：
影响因子：
3.7
通讯作者：
Coonrod S
中科院分区：
文献类型：
--
作者：
Morency E;Anguish L;Coonrod S
我们和其他人最近通过免疫-EM和突变分析证明，两个限制卵母细胞的母性效应基因，PADI6和MATER，部分定位于卵母细胞胞质晶格(CPL)。然而，在这些正在进行的研究中，我们发现，根据卵母细胞和胚胎的处理方式，通过共聚焦免疫荧光(IF)分析对这些因子的定位可能会有很大差异，有时定位模式更均匀地出现在细胞质中，而在其他时候则主要出现在皮质中。为了更好地理解这种不同的染色模式，我们测试了一系列IF方案参数，主要改变了初级抗体溶液孵育的时间和温度条件，以及固定方法。通过共聚焦IF整体贴装分析发现，生发泡期卵母细胞在室温下与一抗孵育1h后，PADI6和MATE主要定位于细胞质，而4℃一抗孵育过夜后，这些因子的定位主要局限于皮质。然后，我们对固定/包埋的卵巢切片和带有特定抗体的分离的两细胞胚胎进行了探测，发现在这些条件下，PADI6和MATER再次主要定位于细胞质，尽管这些因子的染色在两细胞阶段略有皮质。综上所述，我们的结果表明，共聚焦IF对CPL相关蛋白的定位特别受加工条件的影响。此外，根据我们目前的观察，PADI6和MATER似乎主要分布在细胞质中，而不是卵母细胞的皮质下。
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.
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DOI：
10.1002/mrd.1120150408
发表时间：
1986-01-01
期刊：
GAMETE RESEARCH
影响因子：
--
作者：
VISHWANATH, R;SWAN, MA;WHITE, IG
通讯作者：
WHITE, IG
影响因子：
4.8
作者：
Tong, ZB;Nelson, LM
通讯作者：
Nelson, LM
DOI：
10.1073/pnas.0900519106
发表时间：
2009-05-05
影响因子：
11.1
作者：
Zheng, Ping;Dean, Jurrien
通讯作者：
Dean, Jurrien
影响因子：
4.6
作者：
Ohsugi, Mami;Zheng, Ping;Dean, Jurrien
通讯作者：
Dean, Jurrien
DOI：
10.1242/dev.016329
发表时间：
2008-08
期刊：
Development (Cambridge, England)
影响因子：
--
作者：
Yurttas P;Vitale AM;Fitzhenry RJ;Cohen-Gould L;Wu W;Gossen JA;Coonrod SA
通讯作者：
Coonrod SA