| 摘要: |
| 自体荧光是生物体内部组织成分在吸收光时自然发出的荧光，会影响人工标记荧光的有效识别，严重干扰对抗体标记的目的蛋白荧光的观察及数据分析，在珊瑚冰冻切片免疫荧光标记研究中尤为突显。为更有效地去除珊瑚组织切片中的自体荧光，本研究以共生模式种花伞软珊瑚（Xenia sp.）为研究对象，首次尝试在花伞软珊瑚冰冻切片免疫荧光染色中，使用一种在小鼠和人体组织切片中常用的TrueBlack Lipofuscin自体荧光淬灭剂，并与已有研究中的酒精梯度脱水法进行比较。结果表明，使用荧光淬灭剂TrueBlack Lipofuscin后，花伞软珊瑚组织切片中的自体荧光强度是传统的酒精梯度脱水法的52.4%，且在Cy5通道下效果最佳。同时，通过珊瑚免疫荧光标记实验表明使用TrueBlack Lipofuscin的实验组特异性荧光信号强度为对照组的205.3%，背景荧光和自体荧光基本去除。 |
| 关键词: 海洋生物学 珊瑚 自体荧光 免疫荧光标记 冰冻切片 荧光淬灭剂 |
| DOI：10.3969/J.ISSN.2095-4972.20220919002 |
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| 基金项目:福建省自然科学基金(2020J01855) |
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| Elimination of coral autofluorescence with fluorescence quenching agent TrueBlack Lipofuscin and its application in immunofluorescence assay |
| LAI Qingna,WANG Lu,CHEN Jianming |
| (Fujian Key Laboratory on Conservation and Sustainable Utilization of Marine Biodiversity, Minjiang University, Fuzhou 350108, China) |
| Abstract: |
| Autofluorescence is the fluorescence naturally emitted by the internal tissue components of organisms when absorbing light, which will affect the effective recognition of artificial labeling fluorescence and seriously interfere with the observation and data analysis of the fluorescence of antibody labeled target protein, especially in the study of immunofluorescence labeling of coral frozen sections. In order to effectively remove the autofluorescence in the coral tissue slices, this study took the symbiotic model Xeniasp. as the research object, and for the first time tried to use a TrueBlack Lipofuscin autofluorescence quenching agent commonly used in mouse and human tissue slices in the immunofluorescence staining of frozen sections of Xeniasp., and compared it with the alcohol gradient dehydration method in existing studies. Results showed that TrueBlack Lipofuscin could more effectively remove the autofluorescence intensity as much as 52.4% compared with the alcohol gradient dehydration method in the tissue slices of Xeniasp., and the effect was the best under Cy5 channel. At the same time, antibody incubation and protein hybridization experiments proved that the use of autofluorescence quenching agent TrueBlack Lipofuscin did not affect coral immunofluorescence labeling. It showed that the specific fluorescence signal intensity was 205.3% of the control group, and the background fluorescence and autofluorescence were basically removed. |
| Key words: marine biology coral autofluorescence immunofluorescence labeling frozen sections autofluorescence quencher |
