# Protocol for iterative indirect immunofluorescence imaging of frozen mouse intestinal tissues

## TL;DR

This paper provides a detailed, cost-effective protocol for imaging frozen mouse intestinal tissues to detect multiple protein markers at single-cell resolution.

## Contribution

The novel contribution is a streamlined, accessible protocol for iterative immunofluorescence imaging of frozen tissues without requiring specialized equipment or conjugated antibodies.

## Key findings

- The protocol allows multiplexed detection of 10 markers in a single tissue section using five imaging rounds.
- A MATLAB-based pipeline enables quantitative single-cell analysis of the imaging data.
- The method is cost-effective and uses standard reagents and equipment.

## Abstract

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

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.