The Gavin Herbert Eye Institute, University of California Irvine, Irvine, California, United States of America.
PLoS One. 2012;7(12):e53245. doi: 10.1371/journal.pone.0053245. Epub 2012 Dec 31.
Current immunofluorescence protocols are limited as they do not provide reliable antibody staining within large tissue volumes (mm(3)) 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 (mm(3)) 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.
当前的免疫荧光方案受到限制，因为它们不能在大组织体积（mm(3)）内提供可靠的抗体染色，并且不能在同一组织中以高分辨率定位和量化多种抗原或细胞群体。为了解决这个限制，我们开发了一种方法，可以以高分辨率（<1 µm）和多种抗原标记对大组织体积（mm(3)）进行三维可视化，用于体积和定量分析。这是通过对连续切片和顺序免疫染色的丁基甲基甲基丙烯酸酯（BMMA）包埋组织进行计算机重建来实现的。使用这种新颖的免疫荧光计算机断层扫描（ICT）方法，我们已经三维重建了包含睑板腺的小鼠下眼睑的一部分，并定位了细胞核（DAPI）、Ki67 和细胞角蛋白 1（CK1），以及进行了胶原的非线性光学（NLO）显微镜成像，分别评估细胞密度、细胞增殖、腺体角化和腺体体积。在同一块组织上进行四次迭代染色后，抗原性得以维持，这表明只要切片保持完整且先前的抗体已成功洗脱，就可以对重建进行免疫染色的抗原数量没有明确限制。BMMA 树脂包埋还保留了转基因蛋白的荧光。我们提出，ICT 可能为单个组织或器官内的多种生物分子提供有价值的高分辨率三维生物图谱，以更好地表征和量化组织结构和功能。
