Imaging the Drosophila retina: zwitterionic buffers PIPES and HEPES induce morphological artifacts in tissue fixation.
Imaging the Drosophila retina: zwitterionic buffers PIPES and HEPES induce morphological artifacts in tissue fixation.
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DOI：
10.1186/s12861-015-0056-y
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发表时间：
2015-02-03
影响因子：
--
通讯作者：
Zelhof AC
中科院分区：
文献类型：
--
作者：
Nie J;Mahato S;Zelhof AC
组织固定对于保存生物结构的形态和细胞学细节以防止死后降解和自溶至关重要。不适当的固定条件可能导致伪影，从而导致免疫荧光或组织学实验中的错误结论。为了解决果蝇感光细胞组织的结构异常，我们开发并利用了实时成像和固定样品的组合，以调查确切的生物起源，并确定报告的结构差异的潜在来源。我们发现，哌嗪-N，N '-双（乙磺酸）（PIPES）和4-（2-羟乙基）-1-哌嗪乙磺酸（HEPES），两种两性离子缓冲液，通常用于组织固定，可以导致严重的管腔和细胞形态缺陷的果蝇蛹和成人视网膜;横纹肌间腔变得扩张和感光细胞的大小显着减少。相应地，管腔蛋白Eyes shut（EYS）的定位模式严重改变。相比之下，在磷酸盐缓冲盐水（PBS）缓冲液中固定的组织导致与活体成像一致的管腔和细胞形态。我们建议，PIPES和HEPES缓冲液应谨慎使用固定检查细胞和细胞外环境之间的相互作用时，特别是在果蝇蛹和成人视网膜的研究。本文的在线版本（doi：10.1186/s12861-015-0056-y）包含补充材料，可供授权用户使用。
Tissue fixation is crucial for preserving the morphology of biological structures and cytological details to prevent postmortem degradation and autolysis. Improper fixation conditions could lead to artifacts and thus incorrect conclusions in immunofluorescence or histology experiments. To resolve reported structural anomalies with respect to Drosophila photoreceptor cell organization we developed and utilized a combination of live imaging and fixed samples to investigate the exact biogenesis and to identify the underlying source for the reported discrepancies in structure. We found that piperazine-N,N’-bis(ethanesulfonic acid) (PIPES) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), two zwitterionic buffers commonly used in tissue fixation, can cause severe lumen and cell morphological defects in Drosophila pupal and adult retina; the inter-rhabdomeral lumen becomes dilated and the photoreceptor cells are significantly reduced in size. Correspondingly, the localization pattern of Eyes shut (EYS), a luminal protein, is severely altered. In contrast, tissues fixed in the phosphate buffered saline (PBS) buffer results in lumen and cell morphologies that are consistent with live imaging. We suggest that PIPES and HEPES buffers should be utilized with caution for fixation when examining the interplay between cells and their extracellular environment, especially in Drosophila pupal and adult retina research. The online version of this article (doi:10.1186/s12861-015-0056-y) contains supplementary material, which is available to authorized users.
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影响因子：
2.9
作者：
GOOD, NE;WINGET, GD;SINGH, RMM
通讯作者：
SINGH, RMM
影响因子：
4.5
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Nie J;Mahato S;Zelhof AC
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Zelhof AC
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4.5
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影响因子：
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影响因子：
64.5
作者：
REINKE, R;KRANTZ, DE;ZIPURSKY, SL
通讯作者：
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