Department of Medical Laboratory Science, Faculty of Medical Sciences, University of Jos.
Haemato-Oncology Group, Department of Molecular and Clinical Cancer Medicine, University of Liverpool, Liverpool, UK.
Appl Immunohistochem Mol Morphol. 2020 Mar;28(3):249-257. doi: 10.1097/PAI.0000000000000718.
The use of immunofluorescence (IF) technique to detect and evaluate expression levels and localization of cellular proteins and other antigens of interest through the antibodies in their cellular or tissue context has become a standard approach among researchers. Optimizing primary antibody concentrations/dilutions is an essential step in the fluorescent antibody staining protocol. The steps in IF staining are similar to those of the immunohistochemistry (IHC) technique. The use of IHC technique to determine the optimal working dilutions of primary antibodies for IF staining of formalin-fixed paraffin-embedded (FFPE) tissues sections can minimize time wasting and cumbersome approach of using direct IF single labeling using variable dilutions of both primary and secondary antibodies. We used IHC staining technique to determine the working dilutions of the respective primary antibodies by staining 3-µm sections of recommended positive FFPE tissue sections using 3 different dilutions of the primary antibodies and an isotype control (used at the highest concentration). Digital images of sections stained were reviewed in ImageScope by a Consultant Pathologist for positivity, intensity, and histologic distribution. We adopted the IHC predetermined optimal dilutions of primary antibodies to CD4, CD8, CD16, CD21, CD56, CD68, CD163, FOXP3, and PD1 to carry out IF staining of FFPE tissue sections. This approach has helped to remove the complexities associated with grappling with 2 unknown to optimize for both the primary and secondary antibodies using IF technique.
免疫荧光（IF）技术通过抗体在细胞或组织环境中检测和评估细胞蛋白和其他感兴趣抗原的表达水平和定位已成为研究人员的标准方法。优化一抗浓度/稀释度是荧光抗体染色方案中的重要步骤。IF 染色的步骤与免疫组织化学（IHC）技术相似。使用 IHC 技术确定用于 IF 染色的福尔马林固定石蜡包埋（FFPE）组织切片的一抗最佳工作稀释度，可以最大限度地减少使用直接 IF 单标记时使用一抗和二抗的不同稀释度所带来的浪费时间和繁琐的方法。我们使用 IHC 染色技术通过使用一抗的 3 种不同稀释度和同种型对照（在最高浓度下使用）对推荐的阳性 FFPE 组织切片的 3-µm 切片进行染色，来确定各自一抗的工作稀释度。由顾问病理学家在 ImageScope 中对染色的切片的数字图像进行审查，以确定其阳性、强度和组织学分布。我们采用了 IHC 预先确定的最佳 CD4、CD8、CD16、CD21、CD56、CD68、CD163、FOXP3 和 PD1 一抗的稀释度来进行 FFPE 组织切片的 IF 染色。这种方法有助于消除与使用 IF 技术同时优化一抗和二抗相关的复杂性。
