Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.
Ludwig Center for Cancer Research at Harvard, Harvard Medical School, Boston, MA, USA.
Nat Protoc. 2019 Oct;14(10):2900-2930. doi: 10.1038/s41596-019-0206-y. Epub 2019 Sep 18.
Multiplexed tissue imaging enables precise, spatially resolved enumeration and characterization of cell types and states in human resection specimens. A growing number of methods applicable to formalin-fixed, paraffin-embedded (FFPE) tissue sections have been described, the majority of which rely on antibodies for antigen detection and mapping. This protocol provides step-by-step procedures for confirming the selectivity and specificity of antibodies used in fluorescence-based tissue imaging and for the construction and validation of antibody panels. Although the protocol is implemented using tissue-based cyclic immunofluorescence (t-CyCIF) as an imaging platform, these antibody-testing methods are broadly applicable. We demonstrate assembly of a 16-antibody panel for enumerating and localizing T cells and B cells, macrophages, and cells expressing immune checkpoint regulators. The protocol is accessible to individuals with experience in microscopy and immunofluorescence; some experience in computation is required for data analysis. A typical 30-antibody dataset for 20 FFPE slides can be generated within 2 weeks.
多重组织成像技术可实现对人类切除标本中细胞类型和状态的精确、空间分辨的计数和特征分析。越来越多适用于福尔马林固定、石蜡包埋（FFPE）组织切片的方法已经被描述，其中大多数依赖于抗体来进行抗原检测和定位。本方案提供了用于确认荧光组织成像中使用的抗体的选择性和特异性的分步程序，以及抗体面板的构建和验证。尽管该方案是使用基于组织的循环免疫荧光（t-CyCIF）作为成像平台来实现的，但这些抗体测试方法具有广泛的适用性。我们展示了用于计数和定位 T 细胞和 B 细胞、巨噬细胞以及表达免疫检查点调节剂的细胞的 16 种抗体组合。该方案适用于具有显微镜和免疫荧光经验的个人；数据分析需要一些计算经验。在 2 周内可以生成针对 20 个 FFPE 载玻片的典型 30 抗体数据集。
