Schubert W
Department of Molecular Neuropathology, University of Heidelberg, Federal Republic of Germany.
Eur J Cell Biol. 1991 Aug;55(2):272-85.
Two different methods for triple immunofluorescence imaging with a confocal laser scanning microscope (CLSM) are described. The methods enable spatial "mapping" of 3 different epitope distribution patterns simultaneously in one tissue section. The key to triple imaging includes: (a) specific immunolabeling with 3 different mouse monoclonal antibodies (mAbs), (b) localization of the antibody binding sites by 3 different dyes, (c) spectral isolation of each dye by using selective band pass or long pass emission filters, (d) computerized imaging of the fluorescences as colored overlays or as selective signals in each optical section through the tissue in the z-direction. Method 1 consists of the combination of fluorescein isothiocyanate (FITC), phycoerythrin R (PE), and Texas Red (TR) as fluorescent markers. These dyes can be imaged by using 488 nm and 543 nm excitation laser lines. In method 2 aminomethyl coumarin acetic acid (AMCA) was combined with FITC and PE. For this application the CLSM was adapted to ultraviolet microscopy that enabled the use of 3 laser lines (364 nm, 488 nm, 543 nm) for excitations. Cryostat sections of diagnostic human muscle biopsies (n = 9) were studied which were normal by ordinary light microscopic examination. Sections were incubated with mAbs specific for: (1) the fast myosin heavy chain (My32); (2) the major histocompatibility class II antigen HLA-DR; (3) the lymph node homing receptor Leu8, and (4) the cell adhesion receptor OKM5 (CD36). By combining these mAbs in triple staining procedures, 3 capillary types and 4 different phenotypes expressed by muscle fibers were identified simultaneously. The mAbs Leu8 and OKM5, widely used as leukocyte typing antibodies in the blood, exhibit hitherto unrecognized specificities for antigens displayed by muscle fibers. At the level of these markers, specific spatial correlations between OKM5 reactive capillaries and both OKM5 reactive and nonreactive muscle fiber types become visible. The presented results provide direct evidence for cellular complexity and novel insight into the immunoanatomical architecture of skeletal muscle. The methods may be of general significance for the construction and quantification of three-dimensional multiparameter "maps" of cells and tissues.
本文描述了两种使用共聚焦激光扫描显微镜（CLSM）进行三重免疫荧光成像的不同方法。这些方法能够在一个组织切片中同时对三种不同表位分布模式进行空间“映射”。三重成像的关键包括：（a）用三种不同的小鼠单克隆抗体（mAb）进行特异性免疫标记；（b）用三种不同的染料定位抗体结合位点；（c）通过使用选择性带通或长通发射滤光片对每种染料进行光谱分离；（d）将荧光以彩色叠加或作为沿z方向穿过组织的每个光学切片中的选择性信号进行计算机成像。方法1包括将异硫氰酸荧光素（FITC）、藻红蛋白R（PE）和德克萨斯红（TR）作为荧光标记物进行组合。这些染料可以使用488nm和543nm激发激光线进行成像。在方法2中，将氨基甲基香豆素乙酸（AMCA）与FITC和PE进行组合。对于此应用，CLSM被改装为紫外显微镜，从而能够使用三条激光线（364nm、488nm、543nm）进行激发。对诊断性人体肌肉活检的低温切片（n = 9）进行了研究，这些切片在普通光学显微镜检查下是正常的。切片与针对以下物质的mAb一起孵育：（1）快速肌球蛋白重链（My32）；（2）主要组织相容性复合体II类抗原HLA - DR；（3）淋巴结归巢受体Leu8；（4）细胞粘附受体OKM5（CD36）。通过在三重染色程序中组合这些mAb，同时鉴定出了三种毛细血管类型和肌肉纤维表达的四种不同表型。广泛用作血液中白细胞分型抗体的mAb Leu8和OKM5，对肌肉纤维所展示的抗原表现出迄今未被认识到的特异性。在这些标记物水平上，OKM5反应性毛细血管与OKM5反应性和非反应性肌肉纤维类型之间的特定空间相关性变得可见。所呈现的结果为细胞复杂性提供了直接证据，并为骨骼肌的免疫解剖结构提供了新的见解。这些方法对于构建和量化细胞与组织的三维多参数“图谱”可能具有普遍意义。
