Emilson A, Scheynius A
Department of Clinical Immunology, Karolinska Hospital, Stockholm, Sweden.
J Histochem Cytochem. 1995 Oct;43(10):993-8. doi: 10.1177/43.10.7560889.
We used confocal laser scanning microscopy to analyze and compare Langerhans cells (LCs) in normal skin of six subjects. Acetone-fixed epidermal sheets and 25-microns vertical skin sections were incubated with fluorescein isothiocyanate-conjugated mouse monoclonal antibodies directed against HLA-DR. An individual threshold setting algorithm compensating for the differences in background fluorescence was applied to identify specific fluorescence. No statistically significant difference was found in the relative volume of epidermal HLA-DR reactivity between epidermal sheets (14 +/- 5%) and vertical skin sections (13 +/- 6%) or in the number of dendrites per HLA-DR+ LCs (7.8 +/- 3.1 and 5.9 +/- 3.1, n = 58, respectively). However, statistically significant higher background intensity was found in vertical sections than in epidermal sheets. Three-dimensional (3D) reconstructions of HLA-DR+ LCs revealed a concentration of HLA-DR to one or a few intracellular vesicles in 42 of 58 analyzed LCs in epidermal sheets and in 18 of 58 analyzed LCs in vertical sections. Direct contact between dendrites from different LCs was not found. The results indicate that both skin forms are suitable for quantitative studies. Owing to less background intensity and larger tissue volume, detailed 3D analysis of LCs is preferably performed on epidermal sheets rather than on vertical sections.
我们使用共聚焦激光扫描显微镜分析并比较了6名受试者正常皮肤中的朗格汉斯细胞（LCs）。将丙酮固定的表皮片和25微米厚的垂直皮肤切片与针对HLA - DR的异硫氰酸荧光素偶联的小鼠单克隆抗体一起孵育。应用一种补偿背景荧光差异的个体阈值设置算法来识别特异性荧光。在表皮片（14±5%）和垂直皮肤切片（13±6%）之间，表皮HLA - DR反应性的相对体积或每个HLA - DR + LC的树突数量（分别为7.8±3.1和5.9±3.1，n = 58）均未发现统计学上的显著差异。然而，在垂直切片中发现背景强度在统计学上显著高于表皮片。对HLA - DR + LC的三维（3D）重建显示，在表皮片分析的58个LC中有42个以及在垂直切片分析的58个LC中有18个的HLA - DR集中在一个或几个细胞内囊泡中。未发现不同LC的树突之间有直接接触。结果表明两种皮肤形式都适用于定量研究。由于背景强度较低且组织体积较大，对LC的详细3D分析最好在表皮片上进行，而不是在垂直切片上进行。
