18 Histochem Cell Biol (2012) 137:11–24 Fig. 3 Selected images documenting the impact of different mount- ing media and illumination conditions on signal detectability and unspeciﬁc staining over time in different cortical areas (for further details see text). (a, a0, b, b0) Mounting of CISH preparations with Aquatex , as recommended, yielded clear signals, illumination was without effect. (c, c0, d, d0) FISH preparations mounted with Aquatex showed rapid ﬂuorescence fading. (e, e0, f, f0, g, g0, h, h0) HS-TSA-FISH/CISH sections mounted with Fluoro-Gel (e, f) and especially NPG (g, h) showed increased unspeciﬁc staining of the CISH reaction product after prolonged illumination particularly using UV-excitation (DAPI-Filter). Scale bar in (h0) is also valid for (a–h) mounting with Fluoro-Gel the NBT/BCIP signal got weaker and started to become blurry. In FISH/CISH preparations mounted with NPG-containing medium, there was no increased unspeciﬁc staining after bright ﬁeld illumination (Fig. 3g, g0) but these preparations displayed very high unspeciﬁc background staining after 20 min illumination for ﬂuorescence excitation at all wavelengths, for instance after UV illumination (Fig. 3h, h0). Therefore, Fluoro-Gel was used for mounting of all sections subjected to FISH, and, sections were analyzed within a few days after preparation. During analysis, single or double ﬂuorescence-labeled neurons were localized at low magniﬁcation ﬁrst and, if applicable, colocalization of ﬂuorescence was brieﬂy evaluated. Then, the CISH signal was recorded in the same area at higher magniﬁcation. Finally, photographs were taken using bright ﬁeld illumi- nation and ﬂuorescence excitation. To avoid increase of 123 unspeciﬁc nuclear staining, the DAPI signal was watched as brieﬂy as possible, and photographs of the DAPI reac- tion were taken at the end of analysis. Detection speciﬁcity and sensitivity for the different mRNA species Test experiments comparing detectability of the reaction products for the different mRNA species in multiple and in parallel single labelings showed that, under optimal con- ditions, detection speciﬁcity and labeling efﬁciency in double labelings were similar to those achieved in parallel single labelings using the same detection protocols for the individual mRNAs (cf. Figs. 1, 2 and 4, 5). Since with LS-TSA-FISH comparatively fewer NPY-positive cells were observed than using MS-, HS-TSA-FISH or CISH (see above), triple labelings were only qualitatively evaluated.