Protocol for iterative indirect immunofluorescence imaging in cultured cells, tissue sections, and metaphase chromosome spreads

• Published in: STAR protocols Vol. 5; no. 3; p. 103190
• Main Authors: Hsu, Jeffrey, Nguyen, Kimberly T., Bujnowska, Magda, Janes, Kevin A., Fallahi-Sichani, Mohammad
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 20.09.2024; Elsevier
• Subjects: Animals; Cancer; Cell Biology; Cells, Cultured; Fluorescent Antibody Technique, Indirect - methods; Humans; Metaphase; Microscopy; Microscopy, Fluorescence - methods; Protocol; Systems biology; Microscopy; Systems biology; Cell Biology; Cancer
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2024.103190

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.1 [Display omitted] •Three variants of an iterative imaging protocol for cellular and subcellular analysis•Highly multiplexed immunofluorescence staining and antibody elution in diverse samples•Streamlined image processing steps for cultured cells, tissue sections, and chromosomes Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.