Description
Cryo-4i is a modified iterative indirect immunofluorescence imaging (4i) protocol for frozen tissues that avoids antibody conjugation and specialized equipment. Using simple iterative elution steps with standard reagents, it is broadly accessible to molecular biology laboratories. A MATLAB-based pipeline enables quantitative single-cell analysis of protein expression, localization, and modifications. Applied to mouse intestine, Cryo-4i requires two days for setup and one day per imaging round; five rounds allow multiplexed detection of 10 markers in a single section.