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

Area of Science:

• Spatial biology
• Cellular and subcellular imaging
• Molecular pathology

Background:

• Generating high-resolution spatial data is crucial for understanding cellular functions and disease mechanisms.
• Existing methods often lack the multiplexing capacity or resolution required for detailed spatial analysis.
• Iterative indirect immunofluorescence (4i) offers a promising approach to overcome these limitations.

Purpose of the Study:

• To present a streamlined protocol for generating highly multiplexed spatial data using iterative indirect immunofluorescence (4i).
• To demonstrate the adaptability of the 4i protocol across diverse biological samples, including cultured cells, FFPE tissues, and chromosome spreads.
• To provide detailed procedures for sample preparation, staining, imaging, elution, and image processing for high-throughput analysis.

Main Methods:

• Iterative indirect immunofluorescence (4i) imaging protocol.
• Sample preparation for fixed cultured cells, FFPE tissue sections, and metaphase chromosome spreads.
• Antibody and DNA staining, immunofluorescence imaging, antibody elution, and image processing.

Main Results:

• A robust protocol for generating highly multiplexed spatial data at cellular and subcellular resolutions.
• Successful application of the 4i protocol across various sample types, addressing specific challenges like autofluorescence and chromosome fragility.
• Streamlined procedures facilitating high-throughput analysis of spatial biological information.

Conclusions:

• The presented 4i protocol provides a versatile and efficient method for high-resolution spatial data generation.
• This technique significantly enhances the capacity for multiplexed imaging in diverse biological contexts.
• The protocol is well-adapted for high-throughput applications, paving the way for deeper insights into cellular and subcellular organization.