Comparison of Multiple Snap Freezing Protocols and Description of Optimal Workflow v1

The goal of this protocol is to evaluate the optimal snap (flash) freezing method for multiplexed tissue imaging. We detail four different methods (see sample preparation) for preparing snap (flash) frozen tissues. We additionally compare the image quality, ease of use, and safety of each method to our preferred method of tissue preservation (fixed frozen with sucrose cryopreservation). To directly compare tissue preservation methods, tissues were collected from one mouse and divided among the five groups. We determined that the optimal protocol for snap frozen tissues is cryogenic freezing with the Seal'N Freeze Box followed by post-fixation of sections using 1% paraformaldehyde for 10 minutes at room temperature. This method was easy to do, froze the tissue quickly without tissue artifacts, and allowed for immunolabeling of antibodies after fixation.