De Waele M, Renmans W, Segers E, De Valck V, Jochmans K, Van Camp B
Department of Hematology, Academic Hospital, Free University of Brussels, Belgium.
J Histochem Cytochem. 1989 Dec;37(12):1855-62. doi: 10.1177/37.12.2584693.
We developed an indirect immunogold-silver staining method for detection of leukocyte cell surface antigens in cell smears. Air-dried and fixed cytocentrifuge preparations or smears of peripheral blood leukocytes were incubated with monoclonal antibodies (MAb) and colloidal gold-labeled secondary antibodies. The preparations were post-fixed and silver enhancement was performed. The smears were counterstained with May-Grunwald-Giemsa and examined in brightfield light microscopy. The morphology of the cells was well preserved. Leukocytes reacting with the MAb showed black granules on their surface membranes. The intense immunostaining and the low background allowed a rapid enumeration of the positive cells. The labeling could be detected with high sensitivity by epipolarization microscopy. This immunogold-silver staining method was used to quantify T- and B-lymphocytes and natural killer cells in buffy coat smears of normal adult blood. These lymphocyte subsets correlated well with those obtained in smears with the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method and with those found by labeling of mononuclear cells in suspension with immunogold-silver staining. This immunogold-silver staining method forms a good alternative to immunoenzyme methods for study of hematologic cells. In addition, it could be a general procedure for detection of cell surface antigens in all kinds of cell smears.
我们开发了一种间接免疫金银染色法，用于检测细胞涂片上的白细胞表面抗原。将空气干燥并固定的细胞离心涂片或外周血白细胞涂片与单克隆抗体（MAb）和胶体金标记的二抗一起孵育。对涂片进行后固定并进行银增强处理。涂片用美蓝-吉姆萨染色并在明视野光学显微镜下检查。细胞形态保存良好。与MAb反应的白细胞在其表面膜上显示黑色颗粒。强烈的免疫染色和低背景使得能够快速计数阳性细胞。通过偏光显微镜可以高灵敏度地检测到标记。这种免疫金银染色法用于定量正常成人血液血沉棕黄层涂片中的T淋巴细胞、B淋巴细胞和自然杀伤细胞。这些淋巴细胞亚群与用碱性磷酸酶-抗碱性磷酸酶（APAAP）法涂片以及用免疫金银染色标记悬浮单核细胞所获得的结果相关性良好。这种免疫金银染色法是研究血液学细胞的免疫酶法的一种很好的替代方法。此外，它可能是检测各种细胞涂片上细胞表面抗原的通用方法。
