Medical Scientist Training Program, University of Virginia, Charlottesville, VA 22908, USA; Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA 22908, USA.
Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA 22908, USA; Department of Biomedical Engineering, University of Virginia, Charlottesville, VA 22908, USA; UVA Comprehensive Cancer Center, University of Virginia, Charlottesville, VA 22908, USA.
STAR Protoc. 2024 Sep 20;5(3):103190. doi: 10.1016/j.xpro.2024.103190. Epub 2024 Jul 12.
We present a protocol to generate highly multiplexed spatial data at cellular and subcellular resolutions using iterative indirect immunofluorescence imaging (4i). We describe streamlined steps for using 4i across fixed cultured cells, formalin-fixed paraffin-embedded (FFPE) tissue sections, and metaphase chromosome spreads. We detail procedures for sample preparation, antibody and DNA staining, immunofluorescence imaging, antibody elution, and image processing. This protocol is adapted for high-throughput analysis of fixed cultured cells and addresses sample-specific challenges such as intrinsic tissue autofluorescence and chromosome fragility. For complete details on the use and execution of this protocol for fixed cultured cells, please refer to Comandante-Lou et al..
我们提出了一种使用迭代间接免疫荧光成像（4i）在细胞和亚细胞分辨率下生成高度多重化空间数据的方案。我们描述了在固定培养细胞、福尔马林固定石蜡包埋（FFPE）组织切片和中期染色体铺片中使用 4i 的简化步骤。我们详细介绍了样品制备、抗体和 DNA 染色、免疫荧光成像、抗体洗脱和图像处理的程序。该方案适用于固定培养细胞的高通量分析，并解决了样本特异性挑战，如固有组织自发荧光和染色体脆弱性。如需详细了解该方案在固定培养细胞中的使用和执行情况，请参考 Comandante-Lou 等人的研究。
