This is the protocol associated with Combined transcriptomic, connectivity, and activity profiling of the medial amygdala using highly amplified multiplexed in situ hybridization (hamFISH) https://doi.org/10.1101/2024.09.14.613026 In the main report we describe the development of hamFISH and its use in spatially profiling neurons in the medial amygdala of mice. This protocol describes how to perform hamFISH on non-fixed mouse brains, with notes of probe design, probe synthesis and sequential imaging included.