Histochem Cell Biol (2012) 137:11–24 17 Fig. 2 Comparison of different FISH protocols for NPY and 5-HT2C mRNA in La. For NPY mRNA, best results were achieved applying high sensitivity (HS)- and moderate sensitivity (MS)-TSA-FISH (a, b). Arrows indicate low-expressing NPY mRNA-positive cells. With low sensitivity (LS)-TSA-FISH, only high-expressing NPY and unequivocal 5-HT1A mRNA detection was only pos- sible using CISH. MS- and LS-TSA-FISH were not tested for 5-HT1A mRNA since based on the ﬁndings for NPY mRNA detection using these different methods (see above), sufﬁcient sensitivity was not expected. Multiple ISH Mounting media and microscopic analysis Mounting media might exert differential effects on diffu- sion or stability of the chromogen and on ﬂuorescence detectability and fading properties of the ﬂuorescent dye. For CISH and FISH preparations, different mounting media are recommended, such as Aquatex and Fluoro- Gel, respectively. Another common mounting medium for ﬂuorescent preparations is a 60% glycerol/PBS mixture containing 1.5% NPG as an anti-fading additive (Valnes and Brandtzaeg 1985). Since in pilot experiments deterio- ration of signal quality and increased deposition of unspeciﬁc signals had been observed in FISH/CISH pre- parations mounted with different media, we conducted a mRNA-positive neurons were detectable (c). For 5-HT2C mRNA, only HS-TSA-FISH was sufﬁciently sensitive (d); using MS-TSA- FISH, signal-to-noise ratio was low (e). Scale bar in a is also valid for b and c, scale bar in d also for (e) systematic study to determine the most suitable mounting medium and identiﬁed possible pitfalls in the analysis procedures by testing the inﬂuence of these media and of different illumination conditions (bright ﬁeld, ﬂuorescence excitation using ﬁlter systems for DAPI, AlexaFluor 488 and Cy3-detection) on signal detectability and unspeciﬁc staining over time. Results for bright ﬁeld and DAPI illumination are shown in Fig. 3. In sections mounted with Aquatex , which provided optimal stability for CISH preparations under all illumination conditions and over time (Fig. 3a–b0), ﬂuorescence of AlexaFluor 488 and DAPI was rapidly bleached under all illumination condi- tions in FISH preparations (Fig. 3c–d0). Fluorescence fad- ing of FISH signals in FISH/CISH preparations was reduced after mounting with Fluoro-Gel (not shown) and there was no increased unspeciﬁc staining after bright ﬁeld illumination (Fig. 3e, e0) or after illumination using ﬁlter systems for AlexaFluor 488 and Cy3-detection (not shown). However, Fluoro-Gel led to increases in unspeciﬁc, especially nuclear staining, particularly in DAPI-stained FISH/CISH preparations after 20 min of UV illumination (DAPI ﬁlter; Fig. 3f, f0). Five weeks after 123