Protocol for iterative indirect immunofluorescence imaging of frozen mouse intestinal tissues

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. • Procedures for sample preparation of frozen mouse intestinal tissues • Steps for iterative immunofluorescence imaging on frozen tissues using antibody elution • Instructions for image analysis of multiple protein markers at single-cell resolution Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.