A novel immunofluorescent computed tomography (ICT) method to localise and quantify multiple antigens in large tissue volumes at high resolution.
A novel immunofluorescent computed tomography (ICT) method to localise and quantify multiple antigens in large tissue volumes at high resolution.
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DOI：
10.1371/journal.pone.0053245
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Jester JV
中科院分区：
文献类型：
--
作者：
Parfitt GJ;Xie Y;Reid KM;Dervillez X;Brown DJ;Jester JV
目前的免疫荧光方案是有限的，因为它们不能在大组织体积（mm3）内提供可靠的抗体染色，并且不能以高分辨率定位和定量相同组织中的多种抗原或细胞群。为了解决这一限制，我们开发了一种方法，以高分辨率（<1 µm）和多抗原标记三维可视化大组织体积（mm3），用于体积和定量分析。这是通过计算机重建连续切片和顺序免疫染色的丁基甲基丙烯酸甲酯（BMMA）包埋组织。使用这种新的免疫荧光计算机断层扫描（ICT）的方法，我们有三维重建的小鼠下眼睑的一部分，包含睑板腺和局部细胞核（DAPI），Ki67和细胞角蛋白1（CK1），以及执行非线性光学（NLO）显微镜成像的胶原蛋白，以评估细胞密度，细胞增殖，腺体角化和腺体体积分别。在相同组织上进行四次迭代染色后，抗原性得以保持，这表明只要切片保持完整并且先前的抗体已成功洗脱，则可以免疫染色用于重建的抗原数量没有明确的限制。BMMA树脂包埋也保留了转基因蛋白的荧光。我们建议，ICT可以提供有价值的高分辨率，三维生物地图的多个生物分子在一个单一的组织或器官，以更好地识别和量化组织的结构和功能。
Current immunofluorescence protocols are limited as they do not provide reliable antibody staining within large tissue volumes (mm3) and cannot localise and quantify multiple antigens or cell populations in the same tissue at high resolution. To address this limitation, we have developed an approach to three-dimensionally visualise large tissue volumes (mm3) at high resolution (<1 µm) and with multiple antigen labelling, for volumetric and quantitative analysis. This is made possible through computer reconstruction of serial sectioned and sequentially immunostained butyl-methyl methacrylate (BMMA) embedded tissue. Using this novel immunofluorescent computed tomography (ICT) approach, we have three-dimensionally reconstructed part of the murine lower eyelid that contains the meibomian gland and localised cell nuclei (DAPI), Ki67 and cytokeratin 1 (CK1), as well as performing non-linear optical (NLO) microscopy imaging of collagen, to assess cell density, cell proliferation, gland keratinisation and gland volume respectively. Antigenicity was maintained after four iterative stains on the same tissue, suggesting that there is no defined limit to the number of antigens that can be immunostained for reconstruction, as long as the sections remain intact and the previous antibody has been successfully eluted. BMMA resin embedding also preserved fluorescence of transgenic proteins. We propose that ICT may provide valuable high resolution, three-dimensional biological maps of multiple biomolecules within a single tissue or organ to better characterise and quantify tissue structure and function.
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影响因子：
--
作者：
Nien, Chyong Jy;Massei, Salina;Lin, Gloria;Nabavi, Cameron;Tao, Jeremiah;Brown, Donald J.;Paugh, Jerry R.;Jester, James V.
通讯作者：
Jester, James V.
影响因子：
4.3
作者：
BASKIN, TI;BUSBY, CH;GUBLER, F
通讯作者：
GUBLER, F
影响因子：
--
作者：
Liu, Shaohui;Li, Jing;Beuerman, Roger W.
通讯作者：
Beuerman, Roger W.
影响因子：
3.4
作者：
Meagher, CK;Liu, HS;Phillips, TE
通讯作者：
Phillips, TE
影响因子：
3.2
作者：
Panchuk-Voloshina, N;Haugland, RP;Haugland, RP
通讯作者：
Haugland, RP