Quenching autofluorescence of insect tissues for in situ detection of endosymbionts
Quenching autofluorescence of insect tissues for in situ detection of endosymbionts
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DOI：
10.1303/aez.2009.281
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发表时间：
2009-05-01
影响因子：
1.3
通讯作者：
Fukatsu, Takema
中科院分区：
文献类型：
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作者：
Koga, Ryuichi;Tsuchida, Tsutomu;Fukatsu, Takema
寡核苷酸探针荧光原位杂交（FISH）靶向16S rRNA是一种强大的技术，用于检测和表征环境样品中的细菌细胞，而无需培养，然而，该技术的一般应用到昆虫内共生体已受到阻碍，在昆虫组织中经常观察到的强烈的自发荧光。在这里，我们描述了一个协议，显着减少过氧化氢（H（2）O（2））处理的昆虫组织的自发荧光，从而细菌内共生体的16S rRNA保持在FISH检测的条件。在各种组织学固定剂中，Carnoy溶液的优点是上级，与广泛使用的基于甲酰胺的固定剂相比，它成功地固定了整个昆虫，并且抑制了昆虫组织的自发荧光。用6%H2O2乙醇溶液和6%H2O2水溶液处理固定的昆虫组织，均能显著降低其自发荧光，前者能使细菌内共生体的16SrRNA保持在FISH可检测的状态，而后者则不能。该协议适用于不同的昆虫，如蚜虫，虱子和蝙蝠苍蝇的内共生体。该方案不仅适用于新鲜的昆虫样品，而且也适用于保存在丙酮中数年的档案昆虫样品。我们提出了一个通用的和强大的协议，用于猝灭昆虫组织的自发荧光FISH检测细菌内共生体，这是潜在的适用于更广泛的生物体的共生体具有相当大的自发荧光。
Oligonucleotide-probed fluorescent in situ hybridization (FISH) targeting 16S rRNA is a powerful technique for detecting and characterizing bacterial cells in environmental samples without cultivation; however, general application of the technique to insect endosymbionts has been hindered by the strong autofluorescence frequently observed in insect tissues. Here we describe a protocol that markedly reduces autofluorescence of insect tissues by hydrogen peroxide (H(2)O(2)) treatment, whereby 16S rRNA of bacterial endosymbionts is kept in a FISH-detectable condition. Among vafious histological fixatives, Carnoy's solution was superior in that whole insects were successfully fixed and autofluorescence of insect tissues was suppressed in comparison with the widely used formaldehyde-based fixatives. Treatment with both alcoholic 6% H(2)O(2) solution and aqueous 6% H(2)O(2) solution markedly reduced autofluorescence of the fixed insect tissues, wherein the former kept 16S rRNA of bacterial endosymbiont in a FISH-detectable condition while the latter failed to do so. The protocol was applicable to endosymbionts of diverse insects such as aphids, lice and bat flies. The protocol was applicable not only to fresh insect samples but also to archival insect samples preserved in acetone for several years. We propose a general and robust protocol for quenching autofluorescence of insect tissues for FISH detection of bacterial endosymbionts, which is potentially applicable to enclosymbionts of a wider range of organisms with considerable autofluorescence.