Histochem Cell Biol (2012) 137:11–24 single CISH; Merck, Darmstadt, Germany) or Fluoro-Gel (for single and double FISH; Science Services, Munich, Germany) and observed with a Zeiss axioscope (Zeiss, Oberkochen, Germany) equipped with bright ﬁeld illumi- nation and appropriate ﬂuorescence ﬁlter systems for AlexaFluor 488, Cy3 and DAPI ﬂuorescence detection. To determine which mounting medium was best suited for FISH/CISH-reacted sections, the stability of the chromo- genic and ﬂuorescence signals under bright ﬁeld and epiﬂuorescence illumination over time was compared after mounting FISH/CISH-reacted sections in Aquatex (Merck, Darmstadt, Germany), Fluoro-Gel (Science Ser- vices, Munich, Germany) and, additionally, using a 60% glycerol/PBS mixture containing 1.5% n-propyl gallate (NPG) as an anti-fading additive (Valnes and Brandtzaeg 1985). Slides were stored until analysis in light-tight boxes at 4 C. Microphotographs were made using a digital Spot camera (Visitron Systems, Puchheim, Germany) and the Spot Advanced software (Diagnostic Instruments, Inc., Sterling Heights, MI, USA). Identiﬁed neurons were only evaluated as reactive for the different mRNA species if the signals were associated with a morphologically recogniz- able (CISH-reactions) and/or DAPI-labeled nucleus (FISH and FISH/CISH-reactions). Results Comparative single labelings Optimal cRNA probe concentration, hybridization tem- perature and conditions for all cRNA probes were deter- mined in CISH single labelings. After appropriate adaptation of these conditions, CISH for all studied mRNA species showed high signal intensity, high signal-to-noise ratio and lack of labeling in sense controls (Fig. 1a, b, e, f, i, j, m, n). The signal distribution patterns in HS-TSA-FISH and corresponding CISH reactions were similar (Fig. 1a, c, e, g, i, k, m, o); however, the signal-to-noise ratio was generally lower due to background labeling (small ﬂuo- rescent spots) of different intensity, as shown in FISH sense controls (Fig. 1, forth column). While for FISH experiments, the choice of hapten in cRNA labeling (Fluorescein or DIG) had no impact on signal distribution and intensity, highest signal-to-noise ratio was achieved using DIG-labeled probes and AP-conjugated anti-DIG Fab fragments in CISH (not shown). Therefore, only this detection system was applied for CISH in multiple label- ings in order to ensure optimal detection sensitivity. CISH detection of NPY mRNA yielded clear signals in all telencephalic regions studied, with a distribution con- sistent with the pattern described in the literature (e.g., Quidt de and Emson 1986). Signal intensity was similar in 15 CISH and HS-TSA-FISH preparations, for instance in numerous neurons of the hilus of the dentate gyrus (DG; Fig. 1a, c). In isocortical areas and cortex-like amygdaloid nuclei such as the lateral nucleus (La), numerous neurons displaying high, moderate and low NPY-mRNA content were detectable (Fig. 2a). Comparison of HS-, MS- and LS-TSA-FISH for NPY mRNA showed that MS-TSA- FISH was only slightly less sensitive than HS-TSA-FISH, detecting numerous NPY mRNA-positive neurons displaying high-to-low mRNA content (Fig. 2b). Using LS-TSA-FISH, comparatively fewer NPY-positive cells were observed, indicating that with this method neurons with comparatively low content of NPY mRNA were below detection limits (Fig. 2c). CISH for 5-HT2C mRNA showed numerous reactive neurons displaying moderate-to-high levels of mRNA for instance in the DG granular cells, in cortical areas and in the amygdala (e.g., La; Fig. 1e), and similar results were observed using HS-TSA-FISH (Figs. 1g, 2d). Again, the results were consistent with the pattern described in the literature for expression of this receptor subtype (e.g., Mengod et al. 1990). Background labeling, particularly using HS-TSA-FISH, appeared somewhat higher than in ISH for NPY mRNA (cf. Fig. 2a, d). This was even more apparent when 5-HT2C mRNA was detected using MS-TSA-FISH, where the signal-to-noise ratio was too low to allow unequivocal identiﬁcation of labeled neurons (Fig. 2e). LS-TSA-FISH was not tested for this cRNA probe. For 5-HT1A mRNA, ISH signals in the CA pyramidal and the granule cells of the DG, which have been shown to express high levels of this receptor mRNA (Miquel et al. 1991), were comparatively strong using CISH (Fig. 1i), and were also recognizable using HS-TSA-FISH (Fig. 1k). However, unspeciﬁc background labeling was signiﬁcantly higher in HS-TSA-FISH compared to CISH. Thus, while CISH showed very little labeling for instance in the hilus of the DG, numerous small ﬂuorescent dots were observed in this region in HS-TSA-FISH preparations (Fig. 1i, k). In other regions such as the amygdala, where 5-HT1A mRNA expression is reported to be comparatively low (Miquel et al. 1991), detection of 5-HT1A antisense probes with CISH was precise and speciﬁc since the signals, although very discrete, were clearly detectable and could be assigned to speciﬁc cells (Fig. 1m). HS-TSA-FISH detection merely showed an increased density of ﬂuores- cent dots in the vicinity of DAPI-stained nuclei compared to the sense controls (Fig. 1o, p). Since these ﬂuorescent dots were frequently quite dispersed, HS-TSA-FISH sig- nals in regions with closely spaced neurons and compara- tively low expression levels of 5-HT1A could often not be assigned to deﬁned cell bodies, preventing unequivocal identiﬁcation of labeling in speciﬁc cells. Thus, satisfactory 123