20 Histochem Cell Biol (2012) 137:11–24 Fig. 5 Triple ISH for a NPY, b 5-HT1A and c 5-HT2C mRNA in the SC. d Shows a merging of the three images with DAPI staining. Large arrows point to a triple-labeled cell, shown at higher magniﬁcation in the inset in (d), smaller arrows to a cell double-labeled for NPY and was much higher than that of double-labeled neurons (Figs. 4d–f, 5a, b, d). HS-TSA-FISH/CISH was also suc- cessfully applied to detect coexpression of 5-HT1A and 5-HT2C mRNA, for instance in the basolateral nucleus of the amygdala (BL; Fig. 4g–i). From these well-established double labelings, we devel- oped a novel variation of triple ISH applying a combination of double TSA-FISH and CISH. Although it is likely that not all NPY-producing neurons were detected in these prepa- rations since the LS-TSA-FISH method was used for NPY mRNA detection, we were able to provide unequivocal proof of coexpression of both 5-HTR mRNAs in NPY mRNA-reactive neurons, e.g., in the SC (Fig. 5). Speciﬁcity 123 5-HT1A mRNA. Large and small arrowheads indicate cells single- labeled for NPY and 5-HT1A mRNA, respectively. Scale bar in (a) is also valid for (b–d) of the mRNA detection in the triple labelings was supported by the fact that in the same sections neurons coexpressing only NPY and 5-HT1A or only NPY and 5-HT2C mRNA (cf. Fig. 4a–c) in addition to single-labeled NPY and 5-HT1A mRNA-reactive neurons were observed (Fig. 5). Discussion In the present study, we developed a protocol combining TSA-FISH with highly sensitive CISH, and succeeded in a comparatively simple, speciﬁc and reproducible detection of coexpression of low- and moderately abundant 5-HTR