Reliable detection of epigenetic histone marks and nuclear proteins in tissue cryosections
Reliable detection of epigenetic histone marks and nuclear proteins in tissue cryosections
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DOI：
10.1007/s10577-012-9318-8
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发表时间：
2012-10-01
影响因子：
2.6
通讯作者：
Solovei, Irina
中科院分区：
文献类型：
--
作者：
Eberhart, Anja;Kimura, Hiroshi;Solovei, Irina
真实组织中的核过程通常与培养细胞中的核过程显着不同。然而，组织切片上的免疫染色需要长时间固定，这会掩盖抗原，并且分别需要抗原修复，以恢复抗原的可及性。这些治疗会影响免疫染色结果并使其解释变得复杂。这个问题对于核抗原尤其重要，核抗原通常对固定和抗原修复都非常敏感。我们通过研究小鼠视网膜组织切片中的几种组蛋白修饰和核蛋白来解决这个问题，其中包含具有常规和独特倒置核的细胞。在后者中，主要染色质类别形成单独的同心壳，这简化了信号分布的评估。我们发现，通常，较长的固定需要较长的抗原修复时间。然而，抗原在这方面存在显着差异，需要个体调整。我们建议采用一种稳健的切片免疫染色程序，即一种可以控制因固定时间和抗原修复持续时间的差异而引起的免疫染色差异的方法，从而可以快速优化免疫染色方案。
Nuclear processes in real tissues often are significantly different from those in cultured cells. However, immunostaining on tissue sections needs long fixation which masks antigens and, respectively, antigen retrieval which restores antigen accessibility. These treatments affect the immunostaining results and complicate their interpretation. The problem is especially significant for nuclear antigens which often are very sensitive to both fixation and antigen retrieval. We targeted this problem by a study of several histone modifications and nuclear proteins in tissue sections of mouse retina which contains cells with both conventional and unique inverted nuclei. In the latter, the main chromatin classes form separate concentric shells which simplifies evaluation of the signal distribution. We show that as a rule, longer fixation demands longer antigen retrieval time. Nevertheless, antigens are remarkably diverse in this respect and need individual adjustment. We suggest a robust procedure for immunostaining on sections, that is, a method that allows controlling the differences in immunostaining caused by differences in fixation time and antigen retrieval duration, so that immunostaining protocol can be quickly optimized.