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Triple immunofluorescence confocal laser scanning microscopy: spatial correlation of novel cellular differentiation
1Department of Molecular Neuropathology, University of Heidelberg, Federal Republic of Germany.
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.
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.