Poly(A) probe HCR RNA-FISH specifically marks pyriform nurse cells in the brown anole lizard ovary.

Hybridization chain reaction RNA-fluorescent in situ hybridization (HCR RNA-FISH) is a powerful and increasingly used method for visualizing gene expression in cells and tissues. A probe set against polyadenylated RNA (poly(A)) is often used as a positive control for RNA integrity and staining quality. While optimizing this technique in the ovary of the brown anole lizard (Anolis sagrei), we found that the poly(A) probe produced a strikingly specific and intense signal in pyriform cells, a specialized lizard-specific nurse cell type. This staining pattern was found in both whole-mount samples and paraffin sections, suggesting that poly(A) signal intensity can serve as a robust molecular marker for this cell type. The specific and robust signal facilitated segmentation of volumetric data to create the first 3D models of pyriform cells. We also observed unusually diffuse DAPI staining in pyriform cell nuclei, distinguishing them from surrounding granulosa cells, pointing to possible differences in chromatin structure or nuclear organization. Together, these findings highlight the potential of poly(A) probes used in HCR RNA-FISH not only as a technical control, but also as a tool to selectively label specific cell types with high transcriptional activity or storage of abundant poly(A) transcripts.