14 solution diluted 1:50 in AP buffer) for 16–20 h. Develop- ment of the enzyme reaction was carried out in the dark and chromogenic signal intensity was controlled by brief microscopic observation at regular intervals until the desired intensity of staining was achieved. Chromogenic reaction was terminated by washing in stop buffer (10 mM Tris, 0.1 mM EDTA, pH 7.5; 2 9 5 min). For single FISH, three different detection protocols were applied: (1) blocked and hybridized sections were rinsed in washing buffer (3 9 5 min), incubated for 2.5 h in POD- conjugated anti-DIG (1:1,000, Roche) or POD-conjugated anti-Fluorescein Fab fragments (1:1,000, Roche) diluted in antibody incubation buffer, rinsed in washing buffer (3 9 5 min), reacted with TSA Biotin working solution (Perkin Elmer) for 30 min, rinsed in washing buffer (3 9 5 min) again and further incubated in the dark with AlexaFluor 488-conjugated streptavidin (Invitrogen; 5 lg/ml washing buffer) for 30 min. (2) Sections incubated with POD-con- jugated Fab fragments as described above were rinsed in washing buffer (3 9 5 min) and reacted with TSA Cy3 working solution (Perkin Elmer) for 7 min. (3) Sections reacted with a Biotin-labeled cRNA probe were incubated for 30 min with POD-conjugated streptavidin (Perkin Elmer, 1:500 in antibody incubation buffer) rinsed in washing buffer (3 9 5 min) and then reacted with TSA Cy3 working solution for 7 min. Since analyses described below showed that these three mRNA detection systems differed in sensitivity [(1) [ (2) [ (3)], we designated these techniques high sensitivity (HS)-TSA-FISH, moderate sensitivity (MS)- TSA-FISH and low sensitivity (LS)-TSA-FISH, respectively. For double FISH, ﬁrst HS-TSA-FISH was applied to detect one of the cRNA probes. Reacted sections were then subjected to a POD block in 0.02 N HCl for 15 min and rinsed in washing buffer (3 9 5 min) followed by MS-TSA- FISH to detect the second cRNA probe. For FISH/CISH, the appropriate combinations of POD- and AP-conjugated Fab-fragments were applied simultaneously. Fluorescent labeling of cRNA was carried out using HS-TSA-FISH, followed by chromogenic detection with NBT/BCIP, which was carried out as described for single CISH. For triple labelings (double FISH/CISH), sections were ﬁrst subjected to LS-TSA-FISH. After POD block, double FISH/CISH was performed as described above. For all variations, except for single CISH, staining of nuclei was carried out for 5 min using 300 nM DAPI (Roche) diluted in washing buffer. Protocol optimization experiments For each cRNA probe optimal hybridization temperature was determined in single CISH labelings. With NPY and 5-HT2C probe, identical results were obtained between 57 and 60 C, whereas 5-HT1A probe delivered optimal and identical results only between 57 and 58 C. Furthermore, 123 Histochem Cell Biol (2012) 137:11–24 different cRNA probe concentrations between 2 and 20 ng/ll were tested at different hybridization temperatures and best results were achieved using 10 ng/ll for rat NPY and rat 5-HT2C and 15 ng/ll for rat 5-HT1A. Also antibody concentrations, speciﬁcity, and incubation times for all probes were optimized in single CISH single labelings. To compare the inﬂuence of the hapten used for probe labeling and its detection method on ISH sensitivity, CISH and HS-TSA-FISH were carried out for each cRNA probe using both Fluorescein- and DIG-labeled cRNA probes. Additionally, HS-TSA-FISH results were compared with MS- and LS-TSA-FISH results if necessary. During the test experiments for multiple labelings, signal intensities for CISH and HS-TSA-FISH were compared to those observed in single labelings to check whether chro- mogen development and signal interfered with FISH signal detectability and vice versa. For these experiments, sections were covered with coverslips in buffer after the different FISH protocols, and the ﬂuorescence signal intensities and distribution were brieﬂy studied. Subsequently, the cover- slips were removed by dipping the slides in washing buffer, and the reaction sequence was continued. During the ﬁnal step of CISH detection, the sections were incubated in NBT/ BCIP solution and regularly observed using brief exposition with low illumination until the signal was comparable to signals detected in parallel single CISH labeling. After chromogen deposition was terminated, the ﬂuorescent sig- nal in the HS-TSA-FISH/CISH double-labeled sections was studied again and compared to the observations before chromogenic reaction and to parallel single HS-TSA- FISH reactions. Experiments indicated that simultaneous hybridization of different cRNA probes at 57–58 C and application of probe concentrations in the range determined for single labelings yielded high sensitivity and signal-to- noise ratio for combined detections of all mRNA species. Controls Probe speciﬁcity was checked by substitution of the respective antisense cRNA probes with an equivalent amount of labeled sense cRNA probe and by omission of cRNA probes. In multiple ISH reactions, cross controls (mix of antisense and sense cRNA probes followed by detection using the double/triple detection protocols) were carried out for all probe combinations and detection pro- tocols and the results were compared with single ISH for the antisense cRNA probes using the appropriate detection protocol for these probes. Mounting and microscopic analysis of reactions After detection procedure and rinsing in washing buffer (2 9 5 min), sections were mounted using Aquatex (for