Hess Michael W, Pfaller Kristian, Hampölz Bernhard, Longato Stefano, Teis David, Flörl Angelika, Gutleben Karin, Huber Lukas A
Division of Histology and Embryology, Innsbruck Medical University, Innsbruck, Austria.
Microsc Res Tech. 2006 Feb;69(2):93-8. doi: 10.1002/jemt.20268.
We describe here a standardized method for histological processing of the Drosophila compound eye. Primary fixation with 2.5% glutaraldehyde, obligatorily supplemented with 0.1% household detergent regularly yielded the best structural preservation, as compared with that of other, more complicated fixation protocols tested. Notably, it proved indispensable not only to cut off the fly's head to facilitate the penetration of the reagents but also to open the chitinous head capsule. For this, we locally pierced the cuticle between the eyes, leaving the head structurally almost intact, a prerequisite for precisely aligning the head for microtomy. We developed a two-step re-embedding procedure allowing for exact and reproducible orientation of the fly heads. Thus, highly comparable series of cross sections through a representative number of ommatidia were obtained. The feasibility of our embedding and sectioning approach is finally demonstrated by three-dimensional reconstructions of the middle segments of the R1, R7, and R8 photoreceptor cells. We present reconstructions from structurally modified ommatidia, as seen after RNAi-mediated depletion of the endosomal adaptor protein p14, and from normal ommatidia corresponding to the wildtype.
我们在此描述一种用于果蝇复眼组织学处理的标准化方法。与测试的其他更复杂的固定方案相比，用2.5%戊二醛进行初次固定，并强制添加0.1%家用洗涤剂，通常能产生最佳的结构保存效果。值得注意的是，事实证明，不仅要切断果蝇头部以促进试剂渗透，而且要打开几丁质头壳，这一点不可或缺。为此，我们在两眼之间局部刺穿角质层，使头部结构几乎保持完整，这是精确对齐头部以便进行切片的先决条件。我们开发了一种两步重新包埋程序，可实现果蝇头部精确且可重复的定向。因此，获得了通过代表性数量小眼的高度可比的系列横截面。我们通过对R1、R7和R8感光细胞中段的三维重建，最终证明了我们的包埋和切片方法的可行性。我们展示了来自结构修饰小眼的重建结果，如RNA干扰介导的内体衔接蛋白p14缺失后所见，以及来自对应野生型的正常小眼的重建结果。
