CLARITY组织透明化技术在蜜蜂肠道组织上的运用

【目的】以西方蜜蜂Apis mellifera工蜂肠道为例探究组织透明化技术——丙烯酰胺交联替换脂质透明硬化成像/免疫染色/原位杂交兼容组织水凝胶(clear lipid-exchanged acrylamide-hybridized rigid imaging/immunostaining/in situ hybridization-compatible tissue-hYdrogel, CLARITY)在昆虫组织上的应用，确定CLARITY与荧光原位杂交(FISH)相结合在昆虫肠道组织透明化中的适用性。【方法】依照CLARITY技术操作程序，用水凝胶固定西方蜜蜂肠道，并以被动方式透明化，再用靶向东方蜜蜂微孢子虫Nosema ceranae 16S rRNA带异硫氰酸荧光素(fluorescein isothiocyanate, FITC)标记和靶向真核细胞18S rRNA带Texas RED标记的寡核苷酸荧光探针进行肠道组织的荧光原位杂交，然后用DAPI(蓝色)进行细胞核复染，通过激光共聚焦显微镜观察透明化的染色组织。【结果】首次成功将西方蜜蜂肠道组织透明化。在激光共聚焦显微镜下，观察到马氏管的原始分布形态，以及东方蜜蜂微孢子虫在中肠末端分布更密集的空间分布特征，并实现了对肠道组织的3D重构。【结论】CLARITY能应用于蜜蜂肠道组织透明化，透明化组织能进行原位杂交和激光共聚焦观察。CLARITY和FISH相结合免除抗体制备和石蜡切片的麻烦，直观展示肠道内部的真实状态，为昆虫生理病理研究提供了一种可靠特异的标记方法。

Application of CLARITY on honey bee gut tissues

【Aim】 This study aims to investigate whether clear lipid-exchanged acrylamide-hybridized rigid imaging/immunostaining/in situ hybridization compatible tissue-hYdrogel (CLARITY) technique is feasible in insect tissue treatments, using guts of Apis mellifera workers as the test samples, and to test the feasibility of CLARITY in combination with fluorescence in situ hybridization (FISH) in the clarification of insect gut tissues. 【Methods】 A. mellifera worker guts were fixed and solidified according to the regular CLARITY protocol and clarified using the passive method. The in situ hybridization of the clarified gut tissues was conducted using species-specific oligonucleotide probes including Nosema ceranae-specific 16S rRNA-targeted oligonucleotide probe labeled with fluorescein isothiocyanate (FITC) and a universal eukaryotic 18S rRNA targeted oligonucleotide probe labeled with Texas RED. The nuclei were counter-stained with DAPI (in blue), and the stained tissues were observed under a confocal laser scanning microscope. 【Results】 For the first time, A. mellifera guts were successfully processed and clarified. The original morphology of Malpighian tubules in the abdomen was observed, and the spatial distribution of Nosema with higher density at the distal part of the midgut was clearly labeled in the 3D scanning using confocal laser scanning microscope. 【Conclusion】 CLARITY can be used in the clarity of honey bee gut tissues, and the clarified gut tissues can be stained by FISH and observed with confocal laser scanning microscopy. CLARITY in combination with FISH provides a reliable and specific labeling method for insect physiological and pathological studies without the troubles of antibody preparation and paraffin sectioning usually required for a similar study.