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Updated: Mar 10, 2026
A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections
Published on: March 30, 2020
Protocol for iterative indirect immunofluorescence imaging of frozen mouse intestinal tissues
Shuji Matsuguchi1, Aiyan Yau1, Ayaka Omori1
1Laboratory for Quantitative Biology of Cell Fate Decision, Center for Biosystems Dynamics Research, RIKEN, 2-2-3, Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan.
Here, we present a protocol for iterative indirect immunofluorescence imaging (4i) of frozen tissues (Cryo-4i). We describe the steps for mouse intestinal sample preparation and antibody elution with standard reagents. Avoiding antibody conjugation or specialized equipment makes this protocol cost-effective and accessible. It requires two days for setup and one day per imaging round; five rounds allow multiplexed detection of 10 markers in a single section. We also detail procedures for using a MATLAB-based pipeline which enables quantitative single-cell analysis.
Here, we present a protocol for iterative indirect immunofluorescence imaging (4i) of frozen tissues (Cryo-4i). We describe the steps for mouse intestinal sample preparation and antibody elution with standard reagents. Avoiding antibody conjugation or specialized equipment makes this protocol cost-effective and accessible. It requires two days for setup and one day per imaging round; five rounds allow multiplexed detection of 10 markers in a single section. We also detail procedures for using a MATLAB-based pipeline which enables quantitative single-cell analysis.
