12 the results prove synthesis of the proteins to be detected, are often limited in their applicability due to the fact that speciﬁcity of many receptor antibodies is questionable (e.g., McDonald and Mascagni 2007), and that the sensitivity of the detection systems is sometimes not sufﬁcient to detect low quantities of receptor proteins in neuronal membranes. Moreover, peptides and receptors are often localized to speciﬁc subcellular compartments (e.g., neuronal somata vs. processes), and thus an existing coexpression might not be identiﬁable using immunolabeling. ISH avoids these problems, since the mRNAs are concentrated in the cell body and speciﬁcity of cRNA probes can be comparatively easily checked. ISH using enzyme-linked detection systems and chromogenic substrates such as nitro blue tetrazolium (NBT) provides the highest sensitivity (Trinh le et al. 2007), and additionally allows monitoring of the chromogen deposition during the enzyme reaction to determine optimal signal-to-noise ratio. Therefore, chromogenic ISH (CISH) protocols can be reliably used for detection of rare mRNAs. However, multiple CISH with, for instance, alkaline-phos- phatase (AP) and horseradish peroxidase (POD)-linked detection systems cannot usually be combined for coex- pression studies since perikaryal colocalization of two or more colored chromogenic reaction products cannot be unequivocally recognized. Thus, multiple ISH using conventional light and ﬂuo- rescence microscopy has to date been carried out, for instance, using radioactively labeled probes combined with CISH, or multiple ﬂuorescence in situ hybridization (FISH) (Barlier et al. 1999; Denkers et al. 2004; Son and Winzer- Serhan 2009). Although the sensitivity of radioactive ISH is high, the method has its limitations speciﬁcally concerning cellular resolution and signal-to-noise ratio. Conventional FISH protocols allow a sufﬁcient detection only of mRNAs expressed at high abundance (Breininger and Baskin 2000). Recently, tyramide signal ampliﬁcation (TSATM) methods have provided FISH protocols yielding satisfactory results for expression and coexpression studies of mRNAs with moderate abundance, for instance peptide coexpression in the central amygdaloid neurons (Reyes et al. 2008). A further increase in the detection sensitivity appears possible by applying TSA and additional ampliﬁ- cation using an enzyme-labeled ﬂuorescent substrate method (Breininger and Baskin 2000). In the course of studies aimed at unraveling interrelations between mono- aminergic afferents and peptidergic neurons in limbic and sensory areas, we conducted a series of experiments to assess the serotonin receptor (5-HTR) mRNA expression of neuropeptide Y (NPY) mRNA-reactive neurons in the rat telencephalon. NPY has been implied in important CNS functions, ranging from the regulation of food intake and sleep to epilepsy and depression (Vezzani et al. 1999; Sperk et al. 2007; Dyzma et al. 2010; Morales-Medina 123 Histochem Cell Biol (2012) 137:11–24 et al. 2010; Kubota et al. 2011). Functionally important interactions between the serotonergic system and telence- phalic NPY neurons have been suggested, such as an increase in NPY mRNA production in the amygdala upon application of serotonin reuptake inhibitors, and appear to be important with respect to stress response or depression- like behavior (e.g., Luo et al. 2008; Christiansen et al. 2011). Application of double TSA-FISH provided satis- factory results for the detection of 5-HTR subtype mRNAs expressed at high-to-moderate levels in NPY mRNA-pro- ducing neurons. However, the method proved to be insuf- ﬁciently sensitive to detect coexpression of rare 5-HTR subtype mRNAs. To solve this problem, the present study was designed to develop ISH protocols suited for speciﬁc and reproducible detection of mRNA species expressed at low and moderate abundance in peptidergic neurons of rat brain. Materials and methods Generation of cRNA probes Primer sequences speciﬁc for rat NPY (Forward: 50-CAAG CTCATTCCTCGCAGA-30; Reverse: 50-AACGACAACA AGGGAAATGG-30; GenBank accession: NM_012614), rat serotonin receptor 1A (5-HT1A, GenBank accession: NM_012585.1; see Albert et al. 1990) and rat serotonin receptor 2C (5-HT2C, GenBank accession: NM_012765.3; see Julius et al. 1988) mRNA were chosen, cDNA frag- ments in a range of 400–910 bp of size were ampliﬁed and cloned in the dual promotor pCR II (Invitrogen, Karlsruhe, Germany; for NPY fragment), the pGEM -T Vector (Promega, Mannheim, Germany; for 5-HT1A fragment) or the BlueScript SK (?) phagemid (Agilent Technologies, Waldbronn, Germany; for 5-HT2C fragment). The identity of the respective cDNA inserts was veriﬁed by sequencing. For the generation of antisense and sense cRNA probes, plasmids were linearized for 2–3 h at 37 C in a water bath using the appropriate restriction endonucleases. Correct product size was documented by analysis of linearized plasmids on an agarose gel [0.8% agarose, 0.005% Gel RedTM (Biotrend, Cologne, Germany) in 19 TAE buffer consisting of 0.02 M Tris, 0.05 M EDTA, pH 8.3]. Line- arized plasmids were puriﬁed with phenol/chloroform extraction (using TE-saturated phenol, Roth, Karlsruhe, Germany) followed by ethanol precipitation at -20 C over night by adding 0.1 Vol 3 M sodium acetate (pH 5.2) and 2.5 Vol ethanol. Plasmids were centrifuged at 4 C for 30 min, pellets were washed once with 80% ethanol, centrifuged at 4 C for 15 min and air-dried. From now, RNase-free environment was maintained up to post- hybridization steps and all solutions were made with 0.05%