Nie Jing, Mahato Simpla, Zelhof Andrew C
Department of Biology, Indiana University, 1001 East Third St, Bloomington, IN, 47405, USA.
Department of Otolaryngology-Head and Neck Surgery, Indiana University School of Medicine, Indianapolis, IN, 46202, USA.
BMC Dev Biol. 2015 Feb 3;15:10. doi: 10.1186/s12861-015-0056-y.
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.
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。不当的固定条件可能导致假象，从而在免疫荧光或组织学实验中得出错误结论。为了解决所报道的关于果蝇光感受器细胞组织的结构异常问题，我们开发并利用了实时成像和固定样本相结合的方法来研究确切的生物发生过程，并确定所报道的结构差异的潜在根源。
我们发现哌嗪-N,N'-双(乙烷磺酸)(PIPES)和4-(2-羟乙基)-1-哌嗪乙烷磺酸(HEPES)这两种组织固定中常用的两性离子缓冲液，可在果蝇蛹期和成虫期视网膜中导致严重的管腔和细胞形态缺陷；横纹间管腔扩张，光感受器细胞大小显著减小。相应地，管腔蛋白Eyes shut (EYS)的定位模式也发生了严重改变。相比之下，用磷酸盐缓冲盐水(PBS)缓冲液固定的组织，其管腔和细胞形态与实时成像结果一致。
我们建议，在研究细胞与其细胞外环境之间的相互作用时，尤其是在果蝇蛹期和成虫期视网膜研究中，使用PIPES和HEPES缓冲液进行固定时应谨慎。
