A straightforward, robust, and reliable protocol (EASI-FISH) that utilizes expansion microscopy and the hybridization chain reaction for multiplexed in situ hybridization in thick slices of the mouse brain has recently been described (Wang et al., 2021). Below details a modified version of the EASI-FISH protocol for adult Drosophila CNS, which includes antibody detection of fluorescent reporters. The protocol also works well for larval CNS and is expected to be applicable to other tissue types.