Histochem Cell Biol (2012) 137:11–24 19 Fig. 4 Coexpression of NPY, 5-HT2C and 5-HT1A mRNA in different brain areas. a MS-TSA-FISH for NPY mRNA and b HS-TSA-FISH for 5-HT2C mRNA in the Me at high (1), moderate (2) and low (3) expression level. In (c) both images are merged with DAPI staining and coexpression of both mRNAs is visible. d HS-TSA-FISH for NPY mRNA and e CISH for 5-HT1A mRNA in the SC. In (f) both images are merged with DAPI staining Results of multiple labelings Double TSA-FISH delivered satisfactorily sensitive label- ing for coexpression studies of NPY and 5-HT2C mRNA in different brain regions applying MS- and HS-TSA-FISH, respectively. Coexpression of 5-HT2C mRNA was found in subpopulations of NPY mRNA-producing neurons of different areas. The relative frequency of 5-HT2C mRNA expressing among the entire population of NPY-producing neurons differed in a region-speciﬁc way. Thus, although systematic quantitative studies were not carried out, it appeared that while most NPY mRNA-reactive neurons of the medial amygdaloid nucleus (Me) showed receptor mRNA-reactivity (Fig. 4a–c), the subpopulation of 5-HT2C coexpressing NPY mRNA-reactive neurons in and coexpression of both mRNAs is visible (arrow). Arrowheads in (d–f) point to single-labeled NPY and 5-HT1A mRNA-positive cells. g CISH for 5-HT1A mRNA and h HS-TSA-FISH for 5-HT2C mRNA in the BL. In (i) both images are merged with DAPI staining and coexpression of both mRNAs is visible (arrows). Scale bars in (a, d, g) are also valid for (b, c), (e, f) and (h, i), respectively cortical areas, for instance the somatosensory cortex (SC), was lower (Fig. 5a–c). Additionally, the method enabled identiﬁcation of different expression levels of the receptor in NPY neurons (Fig. 4a–c). In all regions analyzed, there were additional 5-HT2C mRNA-reactive neurons devoid of NPY mRNA (not shown). Coexpression of 5-HT1A mRNA could not be identiﬁed in NPY mRNA-reactive neurons unequivocally using double TSA-FISH due to the high background labeling in 5-HT1A FISH (see above). Therefore, HS-TSA-FISH/ CISH was carried out for this combination of cRNA probes and yielded clear colocalization results, e.g., in the SC (Figs. 4d–f, 5). Again, 5-HT1A mRNA-reactive neurons represented a subpopulation of NPY mRNA-producing neurons and the number of 5-HT1A mRNA-positive cells 123