Gene expression detection methods in the African turquoise killifish brain

The short-lived African turquoise killifish (Nothobranchius furzeri) is an important emerging model organism for gene expression studies, with limited tools for transcript and protein detection, especially methods that are both cost-effective and high-resolution. Brain tissue is particularly challenging to analyze due to its opacity and structural complexity, making whole-organ imaging techniques valuable. However, various tissue-clearing protocols adapted for N. furzeri are long and require specialized equipment.

To address these limitations for gene expression detection techniques, we optimized cryosection-compatible ISH protocols for mRNA detection and adapted the EZ-clear method for whole-brain protein visualization in N. furzeri. Using Gfap and Dat as test markers, we optimized the colorimetric ISH protocol for detecting mRNA in both thick and thin sections, achieved high signal-to-noise ratios, and confirmed expression in expected brain regions. Additionally, we adapted the EZ-clear protocol for brain tissue clearing. We demonstrate the method's compatibility with immunostaining, showing a possible upregulation in Gfap, alongside endogenous fluorescence preservation of transgenic reporter lines.

Our protocols add to the existing cost-effective and accessible methods for gene and protein visualization in N. furzeri. The cryosection-amenable ISH and adapted EZ-clear protocols expand the methodological toolkit for studying gene expression in this emerging model system.