Expansion-Assisted Hybridization Chain Reaction-smFISH and Immunohistochemistry in Drosophila Brain

Quantitative, spatially resolved analysis of gene expression is essential for assessing cell-type-specific molecular profiles. In the Drosophila visual system, extensive genetic tools open a framework for direct evaluation of both RNA and protein levels in defined neuronal populations. Here, we present a step-by-step protocol that combines expansion-assisted HCR-smFISH (hybridization chain reaction single-molecule fluorescence in situ hybridization) with immunohistochemistry to enable quantitative analysis of cell-type-specific molecular profiles in genetically defined visual system neuronal types. The workflow is optimized for cells labeled with nuclear-localized or membrane-bound markers, allowing measurement of transcript and protein levels in the same neurons. Following tissue expansion, samples are imaged using light-sheet microscopy for rapid volumetric acquisition, with an alternative mounting and imaging workflow demonstrated for standard inverted laser scanning and spinning disc confocal microscopes. We further provide an automated segmentation algorithm that distinguishes nuclear and cytoplasmic transcripts, enabling analyses of transcriptional state and subcellular RNA localization. Practical guidance is provided on experimental parameters and common pitfalls affecting signal quality, tissue integrity, and quantitative performance. Representative applications include validation of cell-type-specific RNA interference by quantifying corresponding changes in RNA and protein levels. By enabling integrated RNA- and protein-level measurements with cell-type specificity, this approach provides a scalable strategy for hypothesis-driven molecular analysis and, in targeted contexts, a practical alternative to single-cell transcriptomic assays. This protocol provides a practical approach for validating cell-type-specific molecular perturbations while preserving the anatomical context of the intact Drosophila brain.