Shah, S. et al. Neuron 92, 342–357 (2016).

In situ hybridization is a trusted method for obtaining high-resolution spatial expression patterns in organ slices, but the traditional one-gene-at-a-time approach is laborious and does not allow correlations to be drawn between multiple transcripts in the same cell. Shah et al. have improved their multiplexed sequential fluorescence in situ hybridization (SeqFISH) approach and applied it to study the mouse hippocampus. They used the temporal barcoding of five fluorophores to study 125 genes with a new, time-efficient correction scheme that minimizes false calls from erroneously imaged probes. The method incorporates the hybridization chain reaction to amplify signal for more robust detection. By imaging these genes simultaneously in nearly 15,000 cells in tissue sections, the researchers uncovered subregions within the hippocampal CA1 and CA3 regions that exhibit distinct transcriptional profiles.