Division of Surgical Oncology, Department of Surgery, Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX 75390.
Centre for Cancer Immunology, Faculty of Medicine, Southampton General Hospital, University of Southampton, Southampton 016 6YD, United Kingdom.
J Immunol. 2019 Jan 1;202(1):292-299. doi: 10.4049/jimmunol.1800878. Epub 2018 Dec 3.
Immune profiling of tissue through multiplex immunohistochemistry is important for the investigation of immune cell dynamics, and it can contribute to disease prognosis and evaluation of treatment response in cancer patients. However, protocols for mouse formalin-fixed, paraffin-embedded tissue have been less successful. Given that formalin fixation and paraffin embedding remains the most common preparation method for processing mouse tissue, this has limited the options to study the immune system and the impact of novel therapeutics in preclinical models. In an attempt to address this, we developed an improved immunohistochemistry protocol with a more effective Ag-retrieval buffer. We also validated 22 Abs specific for mouse immune cell markers to distinguish B cells, T cells, NK cells, macrophages, dendritic cells, and neutrophils. In addition, we designed and tested novel strategies to identify immune cells for which unique Abs are currently not available. Last, in the 4T1 model of breast cancer, we demonstrate the utility of our protocol and Ab panels in the quantitation and spatial distribution of immune cells.
通过多重免疫组织化学对组织进行免疫分析对于研究免疫细胞动力学非常重要，它可以有助于癌症患者的疾病预后和治疗反应评估。然而，针对小鼠福尔马林固定、石蜡包埋组织的方案不太成功。鉴于福尔马林固定和石蜡包埋仍然是处理小鼠组织最常用的制备方法，这限制了研究免疫系统和新型治疗药物在临床前模型中的影响的选择。为了解决这个问题，我们开发了一种改进的免疫组织化学方案，使用更有效的 Ag 回收缓冲液。我们还验证了 22 种针对小鼠免疫细胞标志物的 Abs，以区分 B 细胞、T 细胞、NK 细胞、巨噬细胞、树突状细胞和中性粒细胞。此外，我们设计并测试了新的策略来识别目前尚无独特 Abs 的免疫细胞。最后，在乳腺癌 4T1 模型中，我们展示了我们的方案和 Abs 面板在免疫细胞定量和空间分布中的实用性。
