Lymph nodes (LNs) are essential secondary immune organs where the adaptive immune response is generated against most infections and vaccines. We recently described the use of live ex vivo LN slices to study the dynamics of adaptive immunity. However, when working with reactive lymph nodes from vaccinated animals, the tissues frequently became dislodged from the supportive agarose matrix during slicing, leading to damage that prevented downstream analysis. Because reactive lymph nodes expand into the surrounding adipose tissue, we hypothesized that dislodging was a result of excess lipids on the collagen capsule of the LN, and that a brief wash with a mild detergent would improve LN interaction with the agarose without damaging tissue viability or function. Therefore, we tested the use of digitonin on improving slicing of vaccinated LNs. Prior to embedding, LNs were quickly dipped into a digitonin solution and washed in saline. Lipid droplets were visibly removed by this procedure. A digitonin wash step prior to slicing significantly reduced the loss of LN during slicing from 13 to 75% to 0–25%, without substantial impact on viability. Capture of fluorescent microparticles, uptake and processing of protein antigen, and cytokine secretion in response to a vaccine adjuvant, R848, were all unaffected by the detergent wash. This novel approach will enable ex vivo analysis of the generation of adaptive immune response in LNs in response to vaccinations and other immunotherapies.

淋巴结 (LN) 是重要的次级免疫器官，针对大多数感染和疫苗产生适应性免疫反应。我们最近描述了使用活体外 LN 切片来研究适应性免疫的动态。然而，当处理来自已接种疫苗的动物的反应性淋巴结时，组织经常在切片过程中从支持性琼脂糖基质上脱落，导致损坏，阻碍下游分析。由于反应性淋巴结扩张到周围的脂肪组织，我们假设移位是由于 LN 胶原蛋白囊上脂质过多造成的，并且用温和的清洁剂进行短暂清洗将改善 LN 与琼脂糖的相互作用，而不会损害组织活力或功能。因此，我们测试了洋地黄皂苷在改善接种 LN 切片方面的用途。包埋之前，将 LN 快速浸入洋地黄皂苷溶液中并用盐水清洗。通过该程序明显去除了脂滴。切片前的毛地黄皂苷清洗步骤可将切片过程中 LN 的损失从 13% 至 75% 显着降低至 0-25%，且对活力没有显着影响。荧光微粒的捕获、蛋白质抗原的摄取和加工以及疫苗佐剂 R848 响应的细胞因子分泌均不受洗涤剂洗涤的影响。这种新方法将能够对 LN 中响应疫苗和其他免疫疗法的适应性免疫反应的产生进行离体分析。